Methods for diagnosing and treating cancer by means of the expression status and mutational status of nrf2 and downstream target genes of said gene

ABSTRACT

The invention provides methods of identifying a subject having cancer, such as lung cancer, by analyzing expression levels of one or more NRF2 splice variants or NRF2 target genes. The invention also provides methods of treating cancer in a subject with a NRF2 pathway antagonist, wherein the subject expresses one or more NRF2 splice variants or overexpresses one or more NRF2 target genes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 20, 2018, isnamed 50474-127002_Sequence_Listing_12.20.18_ST25 and is 216,262 bytesin size.

FIELD OF THE INVENTION

The present invention relates generally to methods for diagnosing,treating, and providing prognoses for cancer, e.g., lung cancer.

BACKGROUND OF THE INVENTION

Cancer remains one of the most deadly threats to human health. Lungcancer, in particular, is the primary cause of cancer-related death formen and women in the United States, despite recent advances intherapeutic treatments. The majority of lung cancers are non-small celllung cancers (NSCLC), and most often of either the adenomatous orsquamous subtype. Recent studies have identified patterns of pointmutations that underlie these indications (Imielinski et al. Cell.150(6):1107-1120, 2012), but despite an increasing number of identifiedmutations associated with various cellular pathways, a comprehensiveunderstanding of the nature and influence of these mutations on thesecellular pathways is lacking.

Thus, there is an unmet need in the field to develop effectivediagnostic and therapeutic strategies for cancers, such as lung cancer.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for diagnosing,treating, and providing prognoses for cancer, for example, lung cancer(e.g., non-small cell lung cancer (NSCLC)) and head and neck carcinoma.

In one aspect, the invention features a method of diagnosing a cancer ina subject, the method comprising: (a) determining the expression levelof at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) gene selectedfrom the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM,TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3,ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN,NQO1, and FTL in a sample obtained from the subject; and (b) comparingthe expression level of the at least one gene to a reference expressionlevel of the at least one gene, wherein an increase in the expressionlevel of the at least one gene in the sample relative to the referenceexpression level of the at least one gene identifies a subject having acancer.

In another aspect, the invention features a method of identifying asubject having a cancer that is a NRF2-dependent cancer, the methodcomprising: (a) determining the expression level of at least one (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, or 27) gene selected from the group consistingof AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR,SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH,TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sampleobtained from the subject; (b) comparing the expression level of the atleast one gene to a reference expression level of the at least one gene;and (c) determining if the subject's cancer is a NRF2-dependent cancer,wherein an increase in the expression level of the at least one gene inthe sample relative to the reference expression level of the at leastone gene identifies a subject having a NRF2-dependent cancer. In someembodiments of either of the preceding aspects, the expression level ofat least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) genes selected from thegroup consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16,ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2,AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, andFTL in a sample obtained from the subject is determined. In someembodiments, the expression level of at least three (e.g., 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, or 27) genes selected from the group consisting of AKR1B10, AKR1C2,SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L,AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR,AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sample obtained from thesubject is determined. In some embodiments, the expression level of atleast four (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, or 27) genes selected from the groupconsisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1,KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15,NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in asample obtained from the subject is determined. In some embodiments, theexpression level of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16,ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2,AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, andFTL in a sample obtained from the subject is determined.

In some embodiments, the expression level of one or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21) ofAKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, KYNU, CABYR,SLC7A11, TRIM16L, AKR1C4, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD,TXN, or NQO1 is determined. In some embodiments, the expression level ofone or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of AKR1B10,AKR1C2, ME1, KYNU, CABYR, TRIM16L, AKR1C4, CYP4F11, RSPO3, AKR1B15,NR0B1, and AKR1C3 is determined.

In some embodiments, (a) the expression level of the at least two genesin the sample is an average (e.g., mean or median) of the at least twogenes of the sample; (b) the reference expression level of the at leasttwo genes is an average (e.g., mean or median) of the at least two genesof the reference; and (c) the average (e.g., mean or median) of the atleast two genes of the sample is compared to the average of the at leasttwo genes of the reference.

In some embodiments, the reference expression level is the mean level ofexpression of the at least one gene in a population of subjects. In someembodiments, the population of subjects is a population of subjectssharing a common ethnicity.

In some embodiments, the reference expression level is the mean level ofexpression of the at least one gene in a population of subjects havingcancer (e.g., lung cancer, e.g., non-small cell lung cancer (NSCLC),e.g., squamous NSCLC).

In some embodiments, the expression level is an mRNA expression level.In some embodiments, the mRNA expression level is determined by PCR,RT-PCR, RNA-seq, gene expression profiling, serial analysis of geneexpression, or microarray analysis.

In other embodiments, the expression level is a protein expressionlevel. In some embodiments, the protein expression level is determinedby western blot, immunohistochemistry, or mass spectrometry.

In some embodiments, any of the preceding methods further comprisesdetermining a DNA sequence of NRF2. In some embodiments, the DNAsequence is determined by PCR, exome-seq, microarray analysis, or wholegenome sequencing.

In another aspect, the invention features a method of diagnosing acancer in a subject, the method comprising determining a DNA sequence ofin a sample obtained from the subject, wherein the presence of NRF2 DNAcomprising a deletion of all or a portion of its exon 2 identifies thesubject as having a cancer. In some embodiments, the DNA sequence isdetermined by PCR, exome-seq, microarray analysis, or whole genomesequencing.

In another aspect, the invention features a method of identifying asubject having cancer, the method comprising determining the mRNAexpression level of NRF2 comprising a deletion of all or a portion ofits exon 2 in a sample obtained from the subject, wherein the presenceof NRF2 comprising a deletion of all or a portion of its exon 2identifies the subject as having a cancer. In some embodiments, the mRNAexpression level is determined by PCR, RT-PCR, RNA-seq, gene expressionprofiling, serial analysis of gene expression, or microarray analysis.In some embodiments, the method further comprises determining a DNAsequence of the NRF2. In some embodiments, the DNA sequence isdetermined by PCR, exome-seq, microarray analysis, or whole genomesequencing.

In some embodiments of any of the preceding aspects, the NRF2 furthercomprises a deletion of all or a portion of its exon 3.

In another aspect, the invention features a method of diagnosing acancer in a subject, the method comprising determining the proteinexpression level of NRF2 comprising a deletion of all or a portion ofits Neh2 domain in a sample obtained from the subject, wherein thepresence of NRF2 comprising a deletion of all or a portion of its Neh2domain identifies the subject as having a cancer.

In another aspect, the invention features a method of identifying asubject having cancer, the method comprising determining the proteinexpression level of NRF2 comprising a deletion of all or a portion ofits Neh2 domain in a sample obtained from the subject, wherein thepresence of NRF2 comprising a deletion of all or a portion of its Neh2domain identifies the subject as having a cancer.

In some embodiments of any of the preceding aspects, the NRF2 furthercomprises a deletion in all or a portion of its Neh4 domain. In someembodiments, the protein expression is determined by western blot,immunohistochemistry, or mass spectrometry.

In some embodiments, the method further comprises administering to thesubject a therapeutically effective amount of a NRF2 pathway antagonist.In some embodiments, the method further comprises administering to thesubject a therapeutically effective amount of an anti-cancer agent. Inother embodiments, the method comprises administering an anti-canceragent and a NRF2 pathway antagonist. In some embodiments, theanti-cancer agent and the NRF2 pathway antagonist are co-administered.In other embodiments, the anti-cancer agent and the NRF2 pathwayantagonist are sequentially administered. In some embodiments, theanti-cancer agent is selected from the group consisting of ananti-angiogenic agent, a chemotherapeutic agent, a growth inhibitoryagent, a cytotoxic agent, and an immunotherapy. In some embodiments, theanti-angiogenic agent is a VEGF antagonist. In some embodiments, theNRF2 pathway antagonist is selected from the group consisting of a CREBantagonist, a CREB Binding Protein (CBP) antagonist, a Maf antagonist,an activating transcription factor 4 (ATF4) antagonist, a protein kinaseC (PKC) antagonist, a Jun antagonist, a glucocorticoid receptorantagonist, a UbcM2 antagonist, a HACE1 antagonist, a c-Myc agonist, aSUMO agonist, a KEAP1 agonist, a CUL3 agonist, or a retinoic acidreceptor α (RARα) agonist.

In another aspect, the invention features a method of treating a subjecthaving a cancer, the method comprising administering to the subject atherapeutically effective amount of a NRF2 pathway antagonist, whereinthe expression level of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or27) of the following genes AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM,TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3,ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN,NQO1, and FTL in a sample obtained from the subject has been determinedto be increased relative to a reference expression level of the at leastone gene. In other embodiments, the expression level of at least two(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, or 27) genes selected from the groupconsisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1,KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15,NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in asample obtained from the subject is determined. In other embodiments,the expression level of at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27)genes selected from the group consisting of AKR1B10, AKR1C2, SRXN1,OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4,CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3,TALDO1, PGD, TXN, NQO1, and FTL in a sample obtained from the subject isdetermined. In other embodiments, the expression level of at least four(e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, or 27) genes selected from the group consisting ofAKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR,SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH,TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sampleobtained from the subject is determined. In other embodiments, theexpression level of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16,ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2,AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, andFTL in a sample obtained from the subject is determined.

In some embodiments, the expression level of one or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21) ofAKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, KYNU, CABYR,SLC7A11, TRIM16L, AKR1C4, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD,TXN, or NQO1 is determined. In other embodiments, the expression levelof one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) ofAKR1B10, AKR1C2, ME1, KYNU, CABYR, TRIM16L, AKR1C4, CYP4F11, RSPO3,AKR1B15, NR0B1, and AKR1C3 is determined.

In some embodiments, (a) the expression level of at least two genes inthe sample is an average of the at least two genes of the sample; (b)the reference expression level of the at least two genes is an averageof the at least two genes of the reference; and (c) the average of theat least two genes of the sample is compared to the average of the atleast two genes of the reference. In some embodiments, the referenceexpression level is the mean level of expression of the at least onegene in a population of subjects. In some embodiments, the population ofsubjects is a population of subjects sharing a common ethnicity. In someembodiments, the reference expression level is the mean level ofexpression of the at least one gene in a population of subjects havingcancer.

In some embodiments, the lung cancer is a non-small cell lung cancer(NSCLC), e.g., squamous NSCLC.

In some embodiments, the expression level is an mRNA expression level.In some embodiments, the mRNA expression level is determined by PCR,RT-PCR, RNA-seq, gene expression profiling, serial analysis of geneexpression, or microarray analysis. In some embodiments, the mRNAexpression level is determined by RNA-seq.

In some embodiments, the method further comprises determining a DNAsequence of the NRF2 (e.g., by PCR, exome-seq, microarray analysis, orwhole genome sequencing).

In some embodiments, the expression level is a protein expression level.In some embodiments, the protein expression is determined by westernblot, immunohistochemistry, or mass spectrometry.

In another aspect, the invention features a method of treating a subjecthaving a cancer, the method comprising: (a) determining the mRNAexpression level of NRF2 comprising a deletion of all or a portion ofits exon 2 in a sample obtained from the subject, wherein the presenceof NRF2 mRNA comprising a deletion of all or a portion of its exon 2identifies the subject as having a cancer; and (b) administering to thesubject a therapeutically effective amount of a NRF2 pathway antagonist.

In some embodiments, the mRNA expression is determined by PCR, RT-PCR,RNA-seq, gene expression profiling, serial analysis of gene expression,or microarray analysis. In some embodiments, the mRNA expression isdetermined by RNA-seq. In some embodiments, the method further comprisesdetermining a DNA sequence of the NRF2 (e.g., by PCR, exome-seq,microarray analysis, or whole genome sequencing).

In another aspect, the invention features a method of treating a subjecthaving a cancer, the method comprising: (a) determining a DNA sequenceof NRF2 comprising a deletion of all or a portion of its exon 2 in asample obtained from the subject, wherein the presence of NRF2 DNAcomprising a deletion of all or a portion of its exon 2 identifies thesubject as having a cancer; and (b) administering to the subject atherapeutically effective amount of a NRF2 pathway antagonist. In someembodiments, the DNA sequence is determined by PCR, exome-seq,microarray analysis, or whole genome sequencing. In some embodiments,the NRF2 (e.g., mRNA or DNA) further comprises a deletion in all or aportion of its exon 3.

In another aspect, the invention features a method of treating a subjecthaving a cancer, the method comprising: (a) determining the proteinexpression level of NRF2 comprising a deletion of all or a portion ofits Neh2 in a sample obtained from the subject, wherein the presence ofNRF2 protein comprising a deletion of all or a portion of its Neh2identifies the subject as having a cancer; and (b) administering to thesubject a therapeutically effective amount of a NRF2 pathway antagonist.

In some embodiments, the NRF2 protein further comprises a deletion ofall or a portion of its Neh4 domain. In some embodiments, the proteinexpression is determined by western blot, immunohistochemistry, or massspectrometry. In some embodiments, the method further comprisesdetermining a DNA sequence of the NRF2 (e.g., by PCR, exome-seq,microarray analysis, or whole genome sequencing).

In some embodiments, the method comprises administering to the subject atherapeutically effective amount of an anti-cancer agent. In someembodiments, the anti-cancer agent and the NRF2 pathway antagonist areco-administered. In other embodiments, the anti-cancer agent and theNRF2 pathway antagonist are sequentially administered. In someembodiments, the anti-cancer agent is selected from the group consistingof an anti-angiogenic agent, a chemotherapeutic agent, a growthinhibitory agent, a cytotoxic agent, and an immunotherapy. In someembodiments, the anti-angiogenic agent is a VEGF antagonist. In someembodiments, the NRF2 pathway antagonist is selected from the groupconsisting of a CREB antagonist, a CREB Binding Protein (CBP)antagonist, a Maf antagonist, an activating transcription factor 4(ATF4) antagonist, a protein kinase C (PKC) antagonist, a Junantagonist, a glucocorticoid receptor antagonist, a UbcM2 antagonist, aHACE1 antagonist, a c-Myc agonist, a SUMO agonist, a KEAP1 agonist, aCUL3 agonist, or a retinoic acid receptor α (RARα) agonist.

In some embodiments, the sample obtained from the subject is a tumorsample, e.g., from a biopsy sample. In some embodiments, the sample isobtained from a previously untreated subject. In some embodiments, thesubject has a lung cancer (e.g., non-small cell lung cancer (NSCLC),e.g., squamous NSCLC) or a head and neck cancer (e.g., squamous head andneck cancer).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a plot showing 96 lung cancer cell lines subjected toRNA-seq, exome-seq, and SNP array analysis. Alterations in KRAS, TP53,KEAP1, EGFR, STK11, NFE2L2, and NF1 are shown.

FIG. 1B is a protein sequence representation showing point mutations inthe NFE2L2 (NRF2) gene.

FIG. 1C is a protein sequence representation showing point mutations inthe KEAP1 gene.

FIG. 1D is an image of the crystal structure of the KEAP1/NRF2 peptidecomplex.

FIG. 2A is a volcano plot illustrating the ratios of average expressionlevels for all genes in mutant (n=25) versus wild-type (WT) (n=74) KEAP1NSCLC cell lines and the associated adjusted p-values resulting from thedifferential expression analysis. Significantly differentially expressedgenes (>2-fold, p<0.01) are indicated, and gene sets previouslyidentified as NRF2 targets are identified as black dots.

FIG. 2B is a heatmap showing the results of unsupervised ward clusteringshowing the upregulation of the 27 genes associated with KEAP1 mutationsin NSCLC cell lines.

FIG. 3A is a heatmap showing the results of an unsupervised wardclustering showing that the NSCLC cell line-derived KEAP1 gene signatureclassified 32 of the 40 (80%) KEAP1 mutant lung adenocarcinomas from thecancer genome atlas (TCGA).

FIG. 3B is a heatmap showing the results of an unsupervised wardclustering showing that the NSCLC cell line-derived KEAP1 gene signatureclassifies 19 of 22 (86%) KEAP1 mutant and 27 of 27 (100%) NRF2 mutantlung squamous cell carcinomas from TCGA.

FIG. 4 is a graph showing the relative abundance of protein products ofthe KEAP1 gene signature in mutant (n=6) and WT (n=37) NSCLC cell lines.

FIG. 5 is a heatmap indicating the frequency of recurrent splicealterations seen in 19 tumor indications.

FIG. 6 shows the NRF2 exons and splice junctions predicted from RNA-seqdata. Predicted features consistent with two annotated refGenetranscripts are shown in gray. Identified exon-exon junctionscorresponding to skip of exon 2 (J2, J5) or exon 2+3 (J3, J6) are shownin black and gray, respectively. A heatmap illustrates read evidence forexon-exon junctions (columns) across 482 TCGA lung squamous carcinoma(rows) on an FPKM scale after log 2(x+1) transformation.

FIG. 7 is a schematic depicting the effect of splice alterations inEGFR, NRF2, MET, and CTNNB1 on protein structure. Arrows indicatein-frame deletions as the result of the splice alteration.

FIG. 8A is a Venn diagram illustrating the mutual exclusive occurrenceof NRF2 splice alteration and mutation in KEAP1 or NRF2 in squamousNSCLC.

FIG. 8B is a heatmap showing clustering of squamous NSCLC based on 27candidate NRF2 target genes. Mutation status and NRF2 splice alterationare indicated for each sample.

FIG. 9A is a Venn diagram illustrating the mutual exclusive occurrenceof NRF2 splice alteration and mutation in KEAP1 or NRF2 in head and neckcancers.

FIG. 9B is a heatmap showing clustering of head and neck cancers basedon 27 candidate NRF2 target genes. Mutation status and NRF2 splicealteration are indicated for each sample.

FIG. 10 is a graph showing the presence of junction reads skipping exon2 in KMS-27 and JHH-6 cells, as quantified by RNA-seq.

FIG. 11A is a schematic diagram showing the locations of exons within WTand exon 2-deleted NRF2 (Δe2 NRF2) mRNA, in relation to forward andreverse primers derived from exon 1 and exons 3/4, indicated byright-hand facing and left-hand facing arrows, respectively.

FIG. 11B is a series of agarose gel images showing RNA productsamplified from total RNA of normal leucocytes, JHH-6 cells, and KMS-27cells, by RT-PCR. Regions surrounding NRF2 exon 2 were amplified withthe indicated primers. Fragments from wild-type NRF2, Δe2 NRF2, andprimer dimers are indicated. Bands indicating the presence of Δe2 NRF2RNA are visible in JHH-6 and KMS-27 cells.

FIG. 12A is a graph showing the sequencing results of the PCR productsfrom JHH-6 and KMS-27 cells, indicating the deletion of exon 2 in NRF2.

FIG. 12B shows the nucleic acid and amino acid sequences for Δe2 NRF2.

FIG. 12C shows the nucleic acid and amino acid sequences for wild-typeNRF2. The exon 2 sequence is shaded.

FIG. 13 shows the results of a Western blot experiment indicating therelative expression of phosphorylated NRF2, wild-type NRF2, and Δe2 NRF2by HUH-1, JHH-6, and HuCCT1 cells. Protein lysates from the indicatedcell lines were separated by SDS PAGE. * represents a likelynon-specific band as it is not depleted by NRF2 siRNA transfection.

FIG. 14A shows the results of a Western blot experiment indicating therelative expression of phosphorylated NRF2, wild-type NRF2, and Δe2 NRF2by HUH-1, JHH-6, and HuCCT1 cells in the presence and absence of lambdaphosphatase (λ P'tase). Cells were grown in 6-well dishes and treatedwith 100 μg/ml cyclohexamide (CHX) for the indicated times. The lysateswere either incubated with buffer or 400 units lambda phosphatase for 30min, before separation by SDS PAGE and Western blotting with NRF2antibodies.

FIG. 14B is a graph showing the stability of NRF2 protein expressed byHuCCT1 cells (circles), JHH-6 cells (squares), and HUH1 cells(triangles) in the presence of CHX. Band intensities from the resultsshown in FIG. 14A were quantified and fitted to a one-phase decay curveto obtain protein half-life estimates, which are indicated next to eachcurve. Relative protein expression was taken as a percent of initialconcentration of each cell line.

FIG. 14C shows the results of a Western blot experiment indicating therelative expression of NRF2 and Δe2 NRF2 by HUH-1, JHH-6, and HuCCT1cells after transfection with either siNTC (50 nM) or siKEAP1 (50 nM).Cells were grown in 6-well dishes and treated with 100 μg/mlcyclohexamide (CHX) for the indicated times. The lysates were incubatedwith 400 units lambda phosphatase for 30 minutes, before separation bySDS PAGE and Western blotting with NRF2 antibodies.

FIG. 14D is a graph showing the stability of NRF2 protein expressed byHuCCT1 cells (circles), JHH-6 cells (squares), and HUH1 cells(triangles) in the presence of CHX after transfection with siNTC (solidlines) or siKEAP1 (dashed lines). Band intensities from the resultsshown in FIG. 14C were quantified and fitted to a one-phase decay curveto obtain protein half-life estimates, which are indicated next to eachcurve. Relative protein expression was taken as a percent of initialconcentration of each cell line.

FIG. 15 shows the results of a Western blot experiment indicating theexpression of Δe2 NRF2 by KMS-27 cells. 20 μg lysates from HCC-1354,KMS-27, and HuCCT1 cells were prepared, and for all except HuCCT1treated with λ P'tase. Untreated and treated lysates were then subjectedto SDS PAGE, and NRF2 and actin were detected.

FIG. 16 shows the results of a Western blot experiment indicatingnuclear localization of NRF2. HuCCT1, HUH-1, and JHH-6 cells were grownin 10 cm dishes and partitioned into nuclear and cytosol fractions.Fractions were separated by SDS PAGE and NRF2 was visualized. Nuclearand cytosolic purity was estimated using Hsp90 as a cytosolic marker andHDAC2 as a nuclear marker.

FIG. 17A is a graph showing the expression of the 27 signature NRF2target genes of the KEAP1 gene signature (each displayed on the x-axis)in 16 hepatocellular carcinoma cell lines (represented by black squares,filled gray circles, and open gray circles) using RNA-seq data describedin Klijn et al. (Nat Biotechnol. 33(3):306-312, 2014). Filled graycircles represent mutant KEAP1 liver cancer cell lines, and open graycircles represent the JHH-6 cell line.

FIG. 17B is a graph showing the expression of the 27 signature NRF2target genes of the KEAP1 gene signature (each displayed on the x-axis)in 18 multiple myeloma cell lines (represented by black squares and opengray circles) using RNA-seq data described in Klijn et al. (Nat.Biotechnol. 33(3):306-312, 2014). Open gray circles represent the KMS-27cell line.

FIG. 18A is a bar graph showing the NRF2 target gene score (meanz-scores for the 27 NRF2 target genes determined over the full data set)in the 16 hepatocellular carcinoma cell lines. KEAP1 and NRF2alterations are indicated as filled and outlined boxes, respectively.

FIG. 18B is a bar graph showing the NRF2 target gene score (meanz-scores for the 27 NRF2 target genes determined over the full data set)in the 18 multiple myeloma cell lines. The outlined box indicates a NRF2alteration.

FIG. 19 is a bar graph showing the viability of HUH-1, JHH-6, and HuCCT1cells in the presence or absence of siRNAs targeting NRF2. Cells wereseeded into 96-well plates containing either a non-targeted siRNAcontrol (NTC), or siRNA targeting NRF2 (NRF2). Viability was measured 4days later using CellTiter-Glo. Viability is presented as a percentageof NTC luminescence.

FIG. 20 is a series of bar graphs showing the effect of transfectionreagents on relative NRF2 expression by HUH-1, JHH-6, and HuCCT1 cells.Cells were grown in 6-well dishes and transfected with siRNA targetingNRF2 exon 5 of NRF2. Total RNA was isolated after 48 hours, and NRF2expression was measured using Taqman probes targeting exon 5.

FIG. 21 is a series of bar graphs showing the effect of transfectionreagents on four well-characterized NRF2 target genes, SLC7A11, GCLC,NR0B1, and SGRN, expressed by HUH-1 cells (dark gray shaded bars), JHH-6cells (light gray shaded bars), and HuCCT1 cells (black shaded bars).Cells were grown in 6-well dishes and transfected with siRNA targetingNRF2exon 5 of NRF2, or non-targeted siRNA (NTC). Total RNA was isolatedafter 48 hours, and gene expression was measured using Taqman probestargeting the indicated NRF2 target genes.

FIG. 22 is a series of representative FACS histograms showing the effectof NRF2 targeting siRNA on DNA fragmentation in HUH-1, JHH-6, and HuCCT1cells. Cells were treated with staurosporin as a positive control.

FIG. 23 is a set of immunoblots showing the effect of NRF2 exon 2 andexon 2+3 deletions on KEAP1 interaction. 293 cells were transfected withplasmids expressing FLAG-NRF2, Δe2 FLAG-NRF2, Δe2+3 FLAG-NRF2 orHA-KEAP1. 48 hours after transfection, cells were lysed, and eitherlysates (top gel) or anti-FLAG immunoprecipitations were analyzed byWestern blotting using the indicated antibodies.

FIG. 24A is a set of immunoblots showing the effect of cyclohexamide onNRF2 stability. 293 cells were transfected with the same plasmids asdescribed in FIG. 23 and treated with 100 μg/ml cycloheximide (CHX) forthe indicated times. Cells were lysed and separated by SDS PAGE, andWestern blotted using NRF2 and anti-actin antibodies.

FIG. 24B is a graph showing the stability of truncated NRF2 followingKEAP1 expression over time.

FIG. 25 is a series of bar graphs showing the expression of various NRF2target genes under various transfection conditions. Cells were treatedas in FIGS. 24A-24B but harvested for total RNA, which was used toanalyze the expression of the indicated genes using Taqman RT-PCR.

FIGS. 26A-1 to 26B-2 are a series of graphs showing the mRNA expressionlevels of indicated NRF2 target genes in TCGA squamous NSCLC tumors,plotted according to mutation status of KEAP1 and NRF2. Individualgraphs show mRNA expression levels of NQO1 (FIG. 26A-1), SLC7A11 (FIG.26B-1), KYNU (FIG. 26C-1), FECH (FIG. 26D-1), CABYR (FIG. 26E-1), GCLM(FIG. 26F-1), TXN (FIG. 26G-1), AKR1C4 (FIG. 26H-1), AKR1C3 (FIG.26I-1), TXNRD1 (FIG. 26J-1), SRXN1 (FIG. 26K-1), GPX2 (FIG. 26L-1),AKR1C2 (FIG. 26M-1), OSGIN1 (FIG. 26N-1), TRIM16 (FIG. 26O-1), NR0B1(FIG. 26P-1), GSR (FIG. 26Q-1), AKR1B10 (FIG. 26R-1), TRIM16L (FIG.26S-1), PGD (FIG. 26T-1), ME1 (FIG. 26U-1), FTL (FIG. 26V-1), RSPO3(FIG. 26W-1), CYP4F11 (FIG. 26X-1), UGDH (FIG. 26Y-1), TALDO1 (FIG.26Z-1), ABCC2 (FIG. 26A-2), and AKR1B15 (FIG. 26B-2). Only samples forwhich both exome-seq and RNA-seq data were available were considered.One sample with mutations in both NRF2 and KEAP1 was excluded. Inaddition, samples with evidence for NRF2 copy number changes|log₂(CAN)|>0.5 were excluded.

FIG. 27A is an exome-seq graph showing relative NRF2 exon abundanceacross 808 cancer cell lines, showing a decrease in reads mapping toexon 2.

FIG. 27B is an exome-seq graph showing normalized z-scores for exon readcoverage across 1,218 squamous NSCLC tumors. Eleven tumors showingdecreased read count for exon 2 or exon 2+3 are compared to nearbycontrol regions.

FIG. 28A is a schematic diagram showing the genomic location ofdiscordant read pairs in seven tumors supporting genomic alterationsaffecting NRF2 exon 2 or exon 2+3.

FIG. 28B-1 is a set of graphs showing the copy number analyses ofchromosome 2 showing two tumor samples with NRF2 exon 2 focal deletions.Arrows point to NRF2 exon 2. The log-ratio of target regions are shownin black and control regions are shown in gray.

FIG. 28B-2 is a set of graphs showing the copy number analyses ofchromosome 2 showing two tumor samples with NRF2 exon 2+3 focaldeletions. Arrows point to NRF2 exon 2 and exon 3. The log-ratio oftarget regions are shown in black and control regions are shown in gray.

FIG. 28C is a series of whole-genome sequencing graphs showing thepresence of microdeletions surrounding NRF2 exon 2 in JHH-6 cells,KMS-27 cells, as well as primary tumor and adjacent matched DNA. Thesequences of reads spanning the deletions are shown NRF2NRF2.

FIG. 29 is a series of agarose gel images showing RNA products amplifiedfrom total RNA of select patients with squamous NSCLC. Shown areamplification products from patient #58 tumor tissue, patient #64 tumortissue, patient #63 normal tissue, and patient #63 tumor tissue byRT-PCR. Regions surrounding NRF2 exon 2 were amplified with the primersindicated in FIG. 11A. Fragments from wild-type NRF2 and Δe2 NRF2 areindicated. RT-PCR analysis identified patient #63 as having loss of NRF2exon 2, which was strongly enriched in the tumor compared to theadjacent normal tissue.

FIG. 30 is a graph showing the presence of junction reads skipping exon2 in tumor and normal cells, as quantified by RNA-seq.

FIG. 31 is a histogram of the mutant KEAP1 gene signature score for TCGAsamples from lung squamous carcinoma (LUSC). Dark gray histogramsrepresent KEAP1/NRF2 mutant tumors, light gray histograms represent exon2/3-deleted tumors, and medium gray histograms represent KEAP1/NRF2wild-type tumors. The gene signature score for a given sample wasdetermined by summation of gene expression z-scores over all genes inthe gene signature.

FIG. 32 is a series of histograms of the mutant KEAP1 gene signaturescore for TCGA samples from lung squamous carcinoma (LUSC), lung adenoma(LUAD), and head and neck squamous carcinoma (HNSC). Dark grayhistograms represent KEAP1/NRF2 mutant tumors, light gray histogramsrepresent exon 2/3-deleted tumors, and medium gray histograms representKEAP1/NRF2 wild-type tumors. The gene signature score for a given samplewas determined by summation of gene expression z-scores over all genesin the gene signature.

FIG. 33 is a series of histograms of the mutant KEAP1 gene signaturescore for TCGA samples from lung squamous carcinoma (LUSC), lung adenoma(LUAD), and head and neck squamous carcinoma (HNSC). Dark grayhistograms represent tumor samples, and light gray histograms representnormal samples. The gene signature score for a given sample wasdetermined by summation of gene expression z-scores over all genes inthe gene signature.

FIG. 34 is a series of junction read sequences showing the structure ofthe deletions in JHH-6 cells, KMS-26 cells, and primary tumor,identified by WGS. The DNA sequences of the 3′ end, 5′ end, and junctionread of JHH-6 cells are provided by SEQ ID NOs: 61-63, respectively. TheDNA sequences of the 3′ end, 5′ end, and junction read of KMS-27 cellsare provided by SEQ ID NOs: 64-66, respectively. The DNA sequences ofthe 3′ end, 5′ end, and junction read of primary tumor cells areprovided by SEQ ID NOs: 67-69, respectively.

FIG. 35 is a series of Western blots showing the relative expression ofNRF2. The indicated cell lines were infected with lentivirusesexpressing independent non-target control (NTC) or three independentNRF2 shRNA sequences (sh1, sh2, and sh3) and were incubated for 48 hourswith (+) or without (−) 500 ng/mL doxycycline (dox) following puromycinselection.

FIG. 36 is a graph showing the viability of the cell lines shown in FIG.35 after incubation with or without dox for 7 days. Viability wasmeasured using CellTiter-Glo (CTG) ATP detection. Each circle is theaverage of six technical replicates, and values were normalized to theaverage percent viability of three independent NTCs+dox.

FIG. 37 is a graph showing the viability of cell lines treated with doxvs no dox. Cells were grown for four days and viability measured usingCTG ATP measurement. Significance was calculated using Student's t test.

FIG. 38 is a graph showing the viability of 28 NSCLC cell linesfollowing treatment with NRF2 siRNA relative to NTC treatment. Cells aregrouped by KEAP1 genotype. Significance was calculated using Student's ttest.

FIG. 39 is a Western blot experiment showing the expression of NRF2 inKEAP1 mutant tumors. Mice were implanted with A549 cells expressing NRF2sh10. When tumors reached ˜200 mm³, 1 mg/ml doxycycline or 5% sucrosewas added to the drinking water. After five days, tumor extracts wereblotted for NRF2.

FIG. 40 is a Western blot experiment showing the expression of NRF2 inKEAP1 wild-type tumors. Mice were implanted with H441 cells expressingNRF2 sh10. When tumors reached ˜200 mm³, 1 mg/ml doxycycline or 5%sucrose was added to the drinking water. After five days, tumor extractswere blotted for NRF2.

FIG. 41A is a graph showing the kinetics of tumor volume in miceimplanted with KEAP1 mutant tumors. Mice were implanted with A549 celllines expressing NRF2 sh10. When tumors reached ˜200 mm³, mice wererandomized into groups of 10, and either 1 mg/ml doxycycline or 5%sucrose was added to the drinking water. Tumors were measured over a28-day period. Error bars represent SEM (n=10).

FIG. 41B is a graph showing the kinetics of tumor volume in miceimplanted with KEAP1 wild-type tumors. Mice were implanted with H441cell lines expressing NRF2 sh10. When tumors reached ˜200 mm³, mice wererandomized into groups of 10, and either 1 mg/ml doxycycline or 5%sucrose was added to the drinking water. Tumors were measured over a28-day period. Error bars represent SEM (n=10).

FIG. 42 is a series of bar graphs showing viability of A549 or H441cells in various growth conditions. A549 and H460 cells expressing NTCor NRF2 sh10 shRNAs were plated into either 2D tissue culture treatedplastic dishes or ultra-low attachment (ULA) coated tissue cultureplates. They were then cultured for five days in either environmentaloxygen concentrations or 0.5% oxygen (hypoxia). Cell viability wasassessed by CTG ATP measurements.

FIG. 43 is a series of photographs showing colony formation of KEAP1mutant cell lines (A549, H1437, and H460) and KEAP1 wild-type cell lines(H1048, H441, and Calu6) in soft agar treated with vehicle, 500 ng/mldox, or 1 mM reduced glutathione (GSH). Representative areas of theplate were photographed.

FIG. 44 is a series of bar graphs showing the quantified colonyformation for each cell type and treatment group shown in FIG. 43. Errorbars represent standard deviation from biological triplicate wells.

FIG. 45 is a series of photographs showing A549 colony formation onSCIVAX® micropatterned nanoculture dishes. Cells were photographed afterabout five days in culture in the presence or absence of 500 ng/ml dox.

FIG. 46 is a bar graph showing viability of the cells from FIG. 45,quantified by CTG ATP measurements. The left column of each treatmentgroup represents 1,000-cell cultures, and the right column represents5,000-cell cultures.

FIG. 47 is a series of photographs showing 5,000, 50,000, or 500,000 NTCor NRF2sh10 shRNA expressing A549 cells plated inmethylcellulose-containing tissue culture dishes. Cells werephotographed after ˜10 days of culture in the presence or absence of 500ng/ml doxycycline.

FIG. 48 is a series of photographs showing A549 cells expressingNRF2sh10 shRNA plated into regular tissue culture dishes (top) or softagar (bottom). Cells were treated with either vehicle or 500 ng/mldoxycycline, in the presence or absence of 2 mM N-acetyl cysteine (NAC).Viability in 2D growth was measured after about five days by CTG ATPmeasurement, and photographs of cells in soft agar were taken afterabout ten days of growth.

FIG. 49 is bar graph showing reactive oxygen species (ROS) levels underindicated conditions as measured using 2′,7′-dichlorodihydrofluoresceindiacetate (H2DCF). Error bars represent standard deviation fromtriplicate wells.

FIG. 50 is a Western blot experiment showing the effect on NRF2knockdown on expression of SLC7A11. A549 cells expressing NRF2 sh10 weretreated with vehicle or 500 ng/ml dox for the indicated time points andblotted using SLC7A11 and p-actin antibodies.

FIG. 51 is a bar graph showing cystine uptake by A549 cells expressingNTC1 or NRF2 sh10 over various concentrations of erastin. A549 cellsexpressing NTC1 or NRF2 sh10 were incubated with vehicle or dox for 48hours, then incubated with 0.5 uCi ¹⁴C-Cystine for 20 minutes. Cellswere lysed and intracellular cystine was measured by liquidscintillation counting.

FIG. 52 is a bar graph showing glutathione (GSH) levels in A549 andH1437 cells in response to NRF2 knockdown.

FIG. 53 is a histogram showing increasing ROS levels in response toshNRF2 and/or erastin, as measured by H2DCF.

FIG. 54 is a graph showing viability of A549 cells expressing shNTC orshNFR2 over a dose response of erastin after about four days, asmeasured using CTG ATP measurements.

FIG. 55A is a graph showing the IC₅₀ of erastin on KEAP1 wild-type celllines versus KEAP1 mutant cell lines, derived from a dose response graphas shown in FIG. 54.

FIG. 55B is a graph showing the viability of KEAP1 wild-type cell linesversus KEAP1 mutant cell lines in response to erastin, as area under thecurve of a dose response graph as shown in FIG. 54.

FIG. 56A is a graph showing the IC₅₀ of the glutaminase inhibitor BPTESon KEAP1 wild-type cell lines versus KEAP1 mutant cell lines.

FIG. 56B is a graph showing the viability of KEAP1 wild-type cell linesversus KEAP1 mutant cell lines in response to the glutaminase inhibitorBPTES.

FIG. 57A is a graph showing the IC₅₀ of the glutathione synthaseinhibitor buthionine sylphoximine (BSO) on KEAP1 wild-type cell linesversus KEAP1 mutant cell lines.

FIG. 57B is a graph showing the viability of KEAP1 wild-type cell linesversus KEAP1 mutant cell lines in response to BSO.

FIG. 58 is a scatterplot showing average gRNA expression per indicatedgene in KEAP1 mutant NSCLC cells grown for 15 days in a 3Dmethylcellulose culture versus a 2D plastic tissue culture dish. A549cells were infected with lentivirus (0.3 MOI at 1000× coverage)expressing a gRNA library comprising 481 NRF2/KEAP1 target genes and 37control genes. Puromycin-resistant cells were then plated into 2Dplastic tissue culture dishes or grown in methyl cellulose. Aftervarious time points, cells were collected and gRNAs identified by NextGen sequencing.

FIG. 59 is a scatterplot showing average gRNA expression per indicatedgene in KEAP1 mutant NSCLC cells implanted in nude mice (xeno) versusgrown for 15 days in a 2D plastic tissue culture dish. A549 cells wereinfected with lentivirus (0.3 MOI at 1000× coverage) expressing a gRNAlibrary comprising 481 NRF2/KEAP1 target genes and 37 control genes.Puromycin-resistant cells were then plated into 2D plastic tissueculture dishes or implanted into nude mice. After various time points,cells were collected and gRNAs identified by Next Gen sequencing.

FIG. 60 is a scatterplot showing average gRNA expression per indicatedgene in KEAP1 mutant NSCLC cells implanted in nude mice (xeno) versusgrown for 15 days in a 3D methylcellulose culture. A549 cells wereinfected with lentivirus (0.3 MOI at 1000× coverage) expressing a gRNAlibrary comprising 481 NRF2/KEAP1 target genes and 37 control genes.Puromycin-resistant cells were then grown in methyl cellulose orimplanted into nude mice. After various time points, cells werecollected and gRNAs identified by Next Gen sequencing.

FIG. 61 is a graph showing kinetics of A549 xenograft tumor volume inresponse to treatment with the Erb2 antibody YW57.88.5.

FIG. 62 is a series of photographs showing colony formation of KEAP1mutant cell lines and KEAP1 wild-type cell lines grown in soft agar(anchorage independent conditions) in the presence of IGF1R inhibitorslinsitinib and NVP-AEW541, and in the presence or absence ofglutathione.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Introduction

The present invention provides diagnostic and accompanying therapeuticmethods for cancer, such as lung cancer (e.g., NSCLC) or head and necksquamous cancer (e.g., HNSC). The invention is based, at least in part,on the discovery that splice variants in NRF2 that remove exon 2 orexons 2+3 result in an unexpected mechanism for conferring NRF2activation in cancers. The NRF2 splice variants result in NRF2activation by a mutually exclusive mechanism from mutations in KEAP1 orNRF2, yet result in a similar NRF2 target gene expression profile. Incell lines with microdeletions that result in these NRF2 splicevariants, there is a loss of NRF2-KEAP1 interaction, increased NRF2stabilization, induction of a NRF2 transcriptional response, and NRF2pathway dependency. This occurs in 3-6% of squamous NSCLC and 1-2% ofHNSC and results in a similar activation of NRF2 target genes anddependency on the pathway as KEAP1 mutations.

This discovery is useful for diagnosing a subject suffering from cancer(e.g., by detecting a NRF2 splice variant or by detecting a gene orprotein expression profile consistent with the presence of a NRF2 splicevariant) and for treating a subject according to such a diagnosis (e.g.,by administering a therapeutically effective amount of a NRF2 pathwayantagonist, e.g., a cAMP Responsive Element Binding Protein (CREB)Binding Protein (CBP) inhibitor).

II. Definitions

The terms “diagnose,” “diagnosing,” or “diagnosis” are used herein torefer to the identification or classification of a molecular orpathological state, disease or condition (e.g., cancer). For example,“diagnosis” may refer to identification of a particular type of cancer.“Diagnosis” may also refer to the classification of a particular subtypeof cancer, e.g., by histopathological criteria, or by molecular features(e.g., a subtype characterized by expression of one or a combination ofbiomarkers (e.g., particular genes or proteins encoded by said genes)).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Included in this definition are benign andmalignant cancers as well as dormant tumors or micrometastatses.Examples of cancer include, but are not limited to, carcinoma, lymphoma,blastoma, glioblastoma, sarcoma, and leukemia. Cancers may include, forexample, breast cancer, squamous cell cancer, lung cancer (includingsmall-cell lung cancer, non-small cell lung cancer (NSCLC),adenocarcinoma of the lung, and squamous carcinoma of the lung (e.g.,squamous NSCLC)), various types of head and neck cancer (e.g., HNSC),cancer of the peritoneum, hepatocellular cancer, gastric or stomachcancer (including gastrointestinal cancer), pancreatic cancer, ovariancancer, cervical cancer, liver cancer, bladder cancer, hepatoma, coloncancer, colorectal cancer, endometrial or uterine carcinoma, salivarygland carcinoma, kidney or renal cancer, liver cancer, prostate cancer,vulval cancer, thyroid cancer, and hepatic carcinoma, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL),small lymphocytic (SL) NHL, intermediate grade/follicular NHL,intermediate grade diffuse NHL, high grade immunoblastic NHL, high gradelymphoblastic NHL, high grade small non-cleaved cell NHL, bulky diseaseNHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom'sMacroglobulinemia), chronic lymphocytic leukemia (CLL), acutelymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblasticleukemia, and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.

A “patient” or “subject” herein refers to any single animal (including,e.g., a mammal, such as a dog, a cat, a horse, a rabbit, a zoo animal, acow, a pig, a sheep, a non-human primate, and a human), such as a human,eligible for treatment who is experiencing or has experienced one ormore signs, symptoms, or other indicators of a disease or disorder, suchas a cancer. Intended to be included as a patient are any patientsinvolved in clinical research trials not showing any clinical sign ofdisease, patients involved in epidemiological studies, or patients onceused as controls. The patient may have been previously treated with aNRF2 pathway antagonist or another drug, or not so treated. The patientmay be naive to an additional drug(s) being used when the treatmentherein is started, i.e., the patient may not have been previouslytreated with, for example, a therapy other than a NRF2 pathwayantagonist (e.g., a VEGF antagonist or a PD-1 axis binding antagonist)at “baseline” (i.e., at a set point in time before the administration ofa first dose of a NRF2 pathway antagonist in the treatment methodherein, such as the day of screening the subject before treatment iscommenced). Such “naive” patients or subjects are generally consideredto be candidates for treatment with such additional drug(s).

The terms “level of expression” or “expression level” in general areused interchangeably and generally refer to the amount of a biomarker ina biological sample. “Expression” generally refers to the process bywhich information (e.g., gene-encoded and/or epigenetic information) isconverted into the structures present and operating in the cell.Therefore, as used herein, “expression” may refer to transcription intoa polynucleotide, translation into a polypeptide, or even polynucleotideand/or polypeptide modifications (e.g., posttranslational modificationof a polypeptide). Fragments of the transcribed polynucleotide, thetranslated polypeptide, or polynucleotide and/or polypeptidemodifications (e.g., post-translational modification of a polypeptide)shall also be regarded as expressed whether they originate from atranscript generated by alternative splicing or a degraded transcript,or from a post-translational processing of the polypeptide, e.g., byproteolysis. “Expressed genes” include those that are transcribed into apolynucleotide as mRNA and then translated into a polypeptide, and alsothose that are transcribed into RNA but not translated into apolypeptide (for example, transfer and ribosomal RNAs).

The terms “biomarker” and “marker” are used interchangeably herein torefer to a DNA, RNA, protein, carbohydrate, or glycolipid-basedmolecular marker, the expression or presence of which in a subject's orpatient's sample can be detected by standard methods (or methodsdisclosed herein). Such biomarkers include, but are not limited to, themRNA sequences set forth in Table 1 and encoded proteins thereof.Expression of such a biomarker may be determined to be higher or lowerin a sample obtained from a patient sensitive or responsive to a NRF2pathway antagonist than a reference level (including, e.g., the average(e.g., mean or median) expression level of the biomarker in a samplefrom a group/population of patients, e.g., patients having cancer, andbeing tested for responsiveness to a NRF2 pathway antagonist; the medianexpression level of the biomarker in a sample from a group/population ofpatients, e.g., patients having cancer, and identified as not respondingto NRF2 pathway antagonists; the level in a sample previously obtainedfrom the individual at a prior time; or the level in a sample from apatient who received prior treatment with a NRF2 pathway antagonist in aprimary tumor setting, and who now may be experiencing metastasis).Individuals having an expression level that is greater than or less thanthe reference expression level of at least one gene, such as those setforth in Table 1 can be identified as subjects/patients likely torespond to treatment with a NRF2 pathway antagonist. For example, suchsubjects/patients who exhibit gene expression levels at the most extreme50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% relative to (i.e.,higher or lower than) the reference level (such as the mean level), canbe identified as subjects/patients (e.g., patients having cancer) likelyto respond to treatment with a NRF2 pathway antagonist.

TABLE 1  SEQ ID NO Biomarker  1 ABCC2  2 AKR1B10  3 AKR1B15  4 AKR1C2  5AKR1C3  6 AKR1C4  7 CABYR  8 CYP4F11  9 FECH 10 FTL 11 GCLM 12 GSR 13KYNU 14 ME1 15 NRF2/NFE2L2 16 NQO1 17 NR0B1 18 OSGIN1 19 PGD 20 RSPO3 21SLC7A11 22 SRXN1 23 TALDO1 24 TRIM16 25 TRIM16L 26 TXN 27 TXNRD1 28 UGDH

The term “ABCC2” as used herein, refers to any native ABCC2 (ATP-BindingCassette Sub-Family C, Member 2) from any vertebrate source, includingmammals such as primates (e.g., humans) and rodents (e.g., mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed ABCC2 as well as any form of ABCC2 that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of ABCC2, e.g., splice variants or allelic variants. Thenucleic acid sequence of an exemplary human ABCC2 is set forth in SEQ IDNO: 1. The amino acid sequence of an exemplary protein encoded by humanABCC2 is shown in SEQ ID NO: 33.

The term “AKR1B10” as used herein, refers to any native AKR1B10(Aldo-Keto Reductase Family 1, Member B10) from any vertebrate source,including mammals such as primates (e.g., humans) and rodents (e.g.,mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed AKR1B10 as well as any form of AKR1B10 thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of AKR1B10, e.g., splice variants or allelicvariants. The nucleic acid sequence of an exemplary human AKR1B10 is setforth in SEQ ID NO: 2. The amino acid sequence of an exemplary proteinencoded by human AKR1B10 is shown in SEQ ID NO: 34.

The term “AKR1B15” as used herein, refers to any native AKR1B15(Aldo-Keto Reductase Family 1, Member B15) from any vertebrate source,including mammals such as primates (e.g., humans) and rodents (e.g.,mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed AKR1B15 as well as any form of AKR1B15 thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of AKR1B15, e.g., splice variants or allelicvariants. The nucleic acid sequence of an exemplary human AKR1B15 is setforth in SEQ ID NO: 3. The amino acid sequence of an exemplary proteinencoded by human AKR1B15 is shown in SEQ ID NO: 35.

The term “AKR1C2” as used herein, refers to any native AKR1C2 (Aldo-KetoReductase Family 1, Member C2) from any vertebrate source, includingmammals such as primates (e.g., humans) and rodents (e.g., mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed AKR1C2 as well as any form of AKR1C2 that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of AKR1C2, e.g., splice variants or allelic variants. Thenucleic acid sequence of an exemplary human AKR1C2 is set forth in SEQID NO: 4. The amino acid sequence of an exemplary protein encoded byhuman AKR1C2 is shown in SEQ ID NO: 36.

The term “AKR1C3” as used herein, refers to any native AKR1C3 (Aldo-KetoReductase Family 1, Member C3) from any vertebrate source, includingmammals such as primates (e.g., humans) and rodents (e.g., mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed AKR1C3 as well as any form of AKR1C3 that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of AKR1C3, e.g., splice variants or allelic variants. Thenucleic acid sequence of an exemplary human AKR1C3 is set forth in SEQID NO: 5. The amino acid sequence of an exemplary protein encoded byhuman AKR1C3 is shown in SEQ ID NO: 37.

The term “AKR1C4” as used herein, refers to any native AKR1C4 (Aldo-KetoReductase Family 1, Member C4) from any vertebrate source, includingmammals such as primates (e.g., humans) and rodents (e.g., mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed AKR1C4 as well as any form of AKR1C4 that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of AKR1C4, e.g., splice variants or allelic variants. Thenucleic acid sequence of an exemplary human AKR1C4 is set forth in SEQID NO: 6. The amino acid sequence of an exemplary protein encoded byhuman AKR1C4 is shown in SEQ ID NO: 38.

The term “CABYR” as used herein, refers to any native CABYR (CalciumBinding Tyrosine-(Y)-Phosphorylation Regulated) from any vertebratesource, including mammals such as primates (e.g., humans) and rodents(e.g., mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed CABYR as well as any form of CABYR thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of CABYR, e.g., splice variants or allelic variants.The nucleic acid sequence of an exemplary human CABYR is set forth inSEQ ID NO: 7. The amino acid sequence of an exemplary protein encoded byhuman CABYR is shown in SEQ ID NO: 39.

The term “CYP4F11” as used herein, refers to any native CYP4F11(Cytochrome P450, Family 4, Subfamily F, Polypeptide 11) from anyvertebrate source, including mammals such as primates (e.g., humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed CYP4F11 as well as any form ofCYP4F11 that results from processing in the cell. The term alsoencompasses naturally occurring variants of CYP4F11, e.g., splicevariants or allelic variants. The nucleic acid sequence of an exemplaryhuman CYP4F11 is set forth in SEQ ID NO: 8. The amino acid sequence ofan exemplary protein encoded by human CYP4F11 is shown in SEQ ID NO: 40.

The term “FECH” as used herein, refers to any native FECH(Ferrochelatase) from any vertebrate source, including mammals such asprimates (e.g., humans) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length,” unprocessedFECH as well as any form of FECH that results from processing in thecell. The term also encompasses naturally occurring variants of FECH,e.g., splice variants or allelic variants. The nucleic acid sequence ofan exemplary human FECH is set forth in SEQ ID NO: 9. The amino acidsequence of an exemplary protein encoded by human FECH is shown in SEQID NO: 41.

The term “FTL” as used herein, refers to any native FTL (Ferritin, LightPolypeptide) from any vertebrate source, including mammals such asprimates (e.g., humans) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length,” unprocessed FTLas well as any form of FTL that results from processing in the cell. Theterm also encompasses naturally occurring variants of FTL, e.g., splicevariants or allelic variants. The nucleic acid sequence of an exemplaryhuman FTL is set forth in SEQ ID NO: 10. The amino acid sequence of anexemplary protein encoded by human FTL is shown in SEQ ID NO: 42.

The term “GCLM” as used herein, refers to any native GCLM(Glutamate-Cysteine Ligase, Modifier Subunit) from any vertebratesource, including mammals such as primates (e.g., humans) and rodents(e.g., mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed GCLM as well as any form of GCLM that resultsfrom processing in the cell. The term also encompasses naturallyoccurring variants of GCLM, e.g., splice variants or allelic variants.The nucleic acid sequence of an exemplary human GCLM is set forth in SEQID NO: 11. The amino acid sequence of an exemplary protein encoded byhuman GCLM is shown in SEQ ID NO: 43.

The term “GSR” as used herein, refers to any native GSR (GlutathioneReductase) from any vertebrate source, including mammals such asprimates (e.g., humans) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length,” unprocessed GSRas well as any form of GSR that results from processing in the cell. Theterm also encompasses naturally occurring variants of GSR, e.g., splicevariants or allelic variants. The nucleic acid sequence of an exemplaryhuman GSR is set forth in SEQ ID NO: 12. The amino acid sequence of anexemplary protein encoded by human GSR is shown in SEQ ID NO: 44.

The term “KYNU” as used herein, refers to any native KYNU (Kynureninase)from any vertebrate source, including mammals such as primates (e.g.,humans) and rodents (e.g., mice and rats), unless otherwise indicated.The term encompasses “full-length,” unprocessed KYNU as well as any formof KYNU that results from processing in the cell. The term alsoencompasses naturally occurring variants of KYNU, e.g., splice variantsor allelic variants. The nucleic acid sequence of an exemplary humanKYNU is set forth in SEQ ID NO: 13. The amino acid sequence of anexemplary protein encoded by human KYNU is shown in SEQ ID NO: 45.

The term “ME1” as used herein, refers to any native ME1 (Malic Enzyme 1,NADP(+)-Dependent, Cytosolic) from any vertebrate source, includingmammals such as primates (e.g., humans) and rodents (e.g., mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed ME1 as well as any form of ME1 that results from processingin the cell. The term also encompasses naturally occurring variants ofME1, e.g., splice variants or allelic variants. The nucleic acidsequence of an exemplary human ME1 is set forth in SEQ ID NO: 14. Theamino acid sequence of an exemplary protein encoded by human ME1 isshown in SEQ ID NO: 46.

The term “NFE2L2” or “NRF2” as used herein, refers to any native NFE2L2or NRF2 (Nuclear Factor, Erythroid 2-Like 2) from any vertebrate source,including mammals such as primates (e.g., humans) and rodents (e.g.,mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed NFE2L2 as well as any form of NFE2L2 thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of NFE2L2, e.g., splice variants or allelic variants.The nucleic acid sequence of an exemplary human NFE2L2 is set forth inSEQ ID NO: 15. The amino acid sequence of an exemplary protein encodedby human NFE2L2 is shown in SEQ ID NO: 47.

The term “NQO1” as used herein, refers to any native NQO1 (NAD(P)HDehydrogenase, Quinone 1) from any vertebrate source, including mammalssuch as primates (e.g., humans) and rodents (e.g., mice and rats),unless otherwise indicated. The term encompasses “full-length,”unprocessed NQO1 as well as any form of NQO1 that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of NQO1, e.g., splice variants or allelic variants. The nucleicacid sequence of an exemplary human NQO1 is set forth in SEQ ID NO: 16.The amino acid sequence of an exemplary protein encoded by human NQO1 isshown in SEQ ID NO: 48.

The term “NR0B1” as used herein, refers to any native NR0B1 (NuclearReceptor Subfamily 0, Group B, Member 1) from any vertebrate source,including mammals such as primates (e.g., humans) and rodents (e.g.,mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed NR0B1 as well as any form of NR0B1 thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of NR0B1, e.g., splice variants or allelic variants.The nucleic acid sequence of an exemplary human NR0B1 is set forth inSEQ ID NO: 17. The amino acid sequence of an exemplary protein encodedby human NR0B1 is shown in SEQ ID NO: 49.

The term “OSGIN1” as used herein, refers to any native OSGIN1 (OxidativeStress Induced Growth Inhibitor 1) from any vertebrate source, includingmammals such as primates (e.g., humans) and rodents (e.g., mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed OSGIN1 as well as any form of OSGIN1 that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of OSGIN1, e.g., splice variants or allelic variants. Thenucleic acid sequence of an exemplary human OSGIN1 is set forth in SEQID NO: 18. The amino acid sequence of an exemplary protein encoded byhuman OSGIN1 is shown in SEQ ID NO: 50.

The term “PGD” as used herein, refers to any native PGD(Phosphogluconate Dehydrogenase) from any vertebrate source, includingmammals such as primates (e.g., humans) and rodents (e.g., mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed PGD as well as any form of PGD that results from processingin the cell. The term also encompasses naturally occurring variants ofPGD, e.g., splice variants or allelic variants. The nucleic acidsequence of an exemplary human PGD is set forth in SEQ ID NO: 19. Theamino acid sequence of an exemplary protein encoded by human PGD isshown in SEQ ID NO: 51.

The term “RSPO3” as used herein, refers to any native RSPO3 (R-Spondin3) from any vertebrate source, including mammals such as primates (e.g.,humans) and rodents (e.g., mice and rats), unless otherwise indicated.The term encompasses “full-length,” unprocessed RSPO3 as well as anyform of RSPO3 that results from processing in the cell. The term alsoencompasses naturally occurring variants of RSPO3, e.g., splice variantsor allelic variants. The nucleic acid sequence of an exemplary humanRSPO3 is set forth in SEQ ID NO: 20. The amino acid sequence of anexemplary protein encoded by human RSPO3 is shown in SEQ ID NO: 52.

The term “SLC7A11” as used herein, refers to any native SLC7A11 (SoluteCarrier Family 7 (Anionic Amino Acid Transporter Light Chain,Xc-System), Member 11) from any vertebrate source, including mammalssuch as primates (e.g., humans) and rodents (e.g., mice and rats),unless otherwise indicated. The term encompasses “full-length,”unprocessed SLC7A11 as well as any form of SLC7A11 that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of SLC7A11, e.g., splice variants or allelic variants. Thenucleic acid sequence of an exemplary human SLC7A11 is set forth in SEQID NO: 21. The amino acid sequence of an exemplary protein encoded byhuman SLC7A11 is shown in SEQ ID NO: 53.

The term “SRXN1” as used herein, refers to any native SRXN1(Sulfiredoxin 1) from any vertebrate source, including mammals such asprimates (e.g., humans) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length,” unprocessedSRXN1 as well as any form of SRXN1 that results from processing in thecell. The term also encompasses naturally occurring variants of SRXN1,e.g., splice variants or allelic variants. The nucleic acid sequence ofan exemplary human SRXN1 is set forth in SEQ ID NO: 22. The amino acidsequence of an exemplary protein encoded by human SRXN1 is shown in SEQID NO: 54.

The term “TALDO1” as used herein, refers to any native TALDO1(Transaldolase 1) from any vertebrate source, including mammals such asprimates (e.g., humans) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length,” unprocessedTALDO1 as well as any form of TALDO1 that results from processing in thecell. The term also encompasses naturally occurring variants of TALDO1,e.g., splice variants or allelic variants. The nucleic acid sequence ofan exemplary human TALDO1 is set forth in SEQ ID NO: 23. The amino acidsequence of an exemplary protein encoded by human TALDO1 is shown in SEQID NO: 55.

The term “TRIM16” as used herein, refers to any native TRIM16(Tripartite Motif Containing 16) from any vertebrate source, includingmammals such as primates (e.g., humans) and rodents (e.g., mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed TRIM16 as well as any form of TRIM16 that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of TRIM16, e.g., splice variants or allelic variants. Thenucleic acid sequence of an exemplary human TRIM16 is set forth in SEQID NO: 24. The amino acid sequence of an exemplary protein encoded byhuman TRIM16 is shown in SEQ ID NO: 56.

The term “TRIM16L” as used herein, refers to any native TRIM16L(Tripartite Motif Containing 16-Like) from any vertebrate source,including mammals such as primates (e.g., humans) and rodents (e.g.,mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed TRIM16L as well as any form of TRIM16L thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of TRIM16L, e.g., splice variants or allelicvariants. The nucleic acid sequence of an exemplary human TRIM16L is setforth in SEQ ID NO: 25. The amino acid sequence of an exemplary proteinencoded by human TRIM16L is shown in SEQ ID NO: 57.

The term “TXN” as used herein, refers to any native TXN (Thioredoxin)from any vertebrate source, including mammals such as primates (e.g.,humans) and rodents (e.g., mice and rats), unless otherwise indicated.The term encompasses “full-length,” unprocessed TXN as well as any formof TXN that results from processing in the cell. The term alsoencompasses naturally occurring variants of TXN, e.g., splice variantsor allelic variants. The nucleic acid sequence of an exemplary human TXNis set forth in SEQ ID NO: 26. The amino acid sequence of an exemplaryprotein encoded by human TXN is shown in SEQ ID NO: 58.

The term “TXNRD1” as used herein, refers to any native TXNRD1(Thioredoxin Reductase 1) from any vertebrate source, including mammalssuch as primates (e.g., humans) and rodents (e.g., mice and rats),unless otherwise indicated. The term encompasses “full-length,”unprocessed TXNRD1 as well as any form of TXNRD1 that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of TXNRD1, e.g., splice variants or allelic variants. Thenucleic acid sequence of an exemplary human TXNRD1 is set forth in SEQID NO: 27. The amino acid sequence of an exemplary protein encoded byhuman TXNRD1 is shown in SEQ ID NO: 59.

The term “UGDH” as used herein, refers to any native UGDH (UridineDiphospho (UDP)-Glucose 6-Dehydrogenase) from any vertebrate source,including mammals such as primates (e.g., humans) and rodents (e.g.,mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed UGDH as well as any form of UGDH that resultsfrom processing in the cell. The term also encompasses naturallyoccurring variants of UGDH, e.g., splice variants or allelic variants.The nucleic acid sequence of an exemplary human UGDH is set forth in SEQID NO: 28. The amino acid sequence of an exemplary protein encoded byhuman UGDH is shown in SEQ ID NO: 60.

The terms “sample” and “biological sample” are used interchangeably torefer to any biological sample obtained from an individual includingbody fluids, body tissue (e.g., tumor tissue), cells, or other sources.Body fluids are, e.g., lymph, sera, whole fresh blood, peripheral bloodmononuclear cells, frozen whole blood, plasma (including fresh orfrozen), urine, saliva, semen, synovial fluid and spinal fluid. Samplesalso include breast tissue, renal tissue, colonic tissue, brain tissue,muscle tissue, synovial tissue, skin, hair follicle, bone marrow, andtumor tissue. Methods for obtaining tissue biopsies and body fluids frommammals are well known in the art.

By “tissue sample” or “cell sample” is meant a collection of similarcells obtained from a tissue of a subject or individual. The source ofthe tissue or cell sample may be solid tissue as from a fresh, frozenand/or preserved organ, tissue sample, biopsy, and/or aspirate; blood orany blood constituents such as plasma; bodily fluids such as cerebralspinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid;cells from any time in gestation or development of the subject. Thetissue sample may also be primary or cultured cells or cell lines.Optionally, the tissue or cell sample is obtained from a diseasetissue/organ. The tissue sample may contain compounds which are notnaturally intermixed with the tissue in nature such as preservatives,anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

A “reference sample,” “reference cell,” “reference tissue,” “controlsample,” “control cell,” or “control tissue,” as used herein, refers toa sample, cell, tissue, standard, or level that is used for comparisonpurposes. In one embodiment, a reference sample, reference cell,reference tissue, control sample, control cell, or control tissue isobtained from a healthy and/or non-diseased part of the body (e.g.,tissue or cells) of the same subject or individual. For example, healthyand/or non-diseased cells or tissue adjacent to the diseased cells ortissue (e.g., cells or tissue adjacent to a tumor). In anotherembodiment, a reference sample is obtained from an untreated tissueand/or cell of the body of the same subject or individual. In yetanother embodiment, a reference sample, reference cell, referencetissue, control sample, control cell, or control tissue is obtained froma healthy and/or non-diseased part of the body (e.g., tissues or cells)of an individual who is not the subject or individual. In even anotherembodiment, a reference sample, reference cell, reference tissue,control sample, control cell, or control tissue is obtained from anuntreated tissue and/or cell of the body of an individual who is not thesubject or individual. In another embodiment, a reference sample,reference cell, reference tissue, control sample, control cell, orcontrol tissue is obtained from one or more cell lines (e.g., one ormore normal cell lines).

The phrase “identifying a patient” or “identifies a patient” as usedherein, refers to using the information or data generated relating tothe level of at least one of the genes set forth in Table 1, thepresence of NRF2 mRNA having deletion of all or a portion of its exon 2or exon 2+3, or the presence of NRF2 protein having a deletion of all ora portion of its Neh2 or Neh2+4 in a sample of a patient to identify orselect the patient as more likely to benefit or less likely to benefitfrom a therapy comprising a NRF2 pathway antagonist. The information ordata used or generated may be in any form, written, oral or electronic.In some embodiments, using the information or data generated includescommunicating, presenting, reporting, storing, sending, transferring,supplying, transmitting, dispensing, or combinations thereof. In someembodiments, communicating, presenting, reporting, storing, sending,transferring, supplying, transmitting, dispensing, or combinationsthereof are performed by a computing device, analyzer unit orcombination thereof. In some further embodiments, communicating,presenting, reporting, storing, sending, transferring, supplying,transmitting, dispensing, or combinations thereof are performed by alaboratory or medical professional. In some embodiments, the informationor data includes a comparison of the level of at least one of the genesset forth in Table 1 to a reference level. In some embodiments, theinformation or data includes an indication that at least one of thegenes set forth in Table 1 is present or absent in the sample. In someembodiments, the information or data includes an indication that theNRF2 mRNA has a deletion of all or a portion of its exon 2 or exon 2+3.In some embodiments, the information or data includes an indication thatthe NRF2 protein has a deletion of all or a portion of its Neh2 orNeh2+4. In some embodiments, the information or data includes anindication that the patient is more likely or less likely to respond toa therapy comprising a NRF2 pathway antagonist).

The term “primer” refers to a single-stranded polynucleotide that iscapable of hybridizing to a nucleic acid and allowing polymerization ofa complementary nucleic acid, generally by providing a free 3′—OH group.

As used herein, the term “treatment” (and variations thereof, such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

As used herein, “administering” is meant a method of giving a dosage ofa compound (e.g., a NRF2 pathway antagonist) to a subject. Thecompositions utilized in the methods described herein can beadministered, for example, intravitreally (e.g., by intravitrealinjection), by eye drop, intramuscularly, intravenously, intradermally,percutaneously, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostatically, intrapleurally,intratracheally, intrathecally, intranasally, intravaginally,intrarectally, topically, intratumorally, peritoneally, subcutaneously,subconjunctivally, intravesicularly, mucosally, intrapericardially,intraumbilically, intraocularly, intraorbitally, orally, topically,transdermally, by inhalation, by injection, by implantation, byinfusion, by continuous infusion, by localized perfusion bathing targetcells directly, by catheter, by lavage, in cremes, or in lipidcompositions. The compositions utilized in the methods described hereincan also be administered systemically or locally. The method ofadministration can vary depending on various factors (e.g., the compoundor composition being administered and the severity of the condition,disease, or disorder being treated).

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “anti-neoplastic” refers to a composition useful in treatingcancer comprising at least one active therapeutic agent, e.g.,“anti-cancer agent.” Examples of therapeutic agents (anti-cancer agents)include, but are limited to, e.g., chemotherapeutic agents, growthinhibitory agents, cytotoxic agents, agents used in radiation therapy,anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, andother-agents to treat cancer, such as anti-HER-2 antibodies, anti-CD20antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g.,a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib(TARCEVA™) platelet derived growth factor inhibitors (e.g., GLEEVEC™(Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons,cytokines, antagonists (e.g., neutralizing antibodies) that bind to oneor more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS,APRIL, BCMA or VEGF receptor(s), TRAIL/Apo2, and other bioactive andorganic chemical agents, and the like. Combinations thereof are alsoincluded in the invention.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents include is achemical compound useful in the treatment of cancer. Examples ofchemotherapeutic agents include alkylating agents, such as, for example,temozolomide (TMZ), the imidazotetrazine derivative of the alkylatingagent dacarbazine. Additional examples of chemotherapeutics agentsinclude, e.g., paclitaxel or topotecan or pegylated liposomaldoxorubicin (PLD). Other examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin; bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegal1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf,H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF that reduce cellproliferation and pharmaceutically acceptable salts, acids, orderivatives of any of the above.

The terms “Programmed Death Ligand 1” and “PD-L1” refer herein to anative sequence PD-L1 polypeptide, polypeptide variants, and fragmentsof a native sequence polypeptide and polypeptide variants. The PD-L1polypeptide described herein may be that which is isolated from avariety of sources, such as from human tissue types or from anothersource, or prepared by recombinant or synthetic methods.

The term “PD-L1 axis binding antagonist” refers to a molecule thatinhibits the interaction of a PD-L1 axis binding partner with one ormore of its binding partners, so as to remove T-cell dysfunctionresulting from signaling on the PD-1 signaling axis, with a result beingrestored or enhanced T-cell function. As used herein, a PD-L1 axisbinding antagonist includes a PD-L1 binding antagonist and a PD-1binding antagonist as well as molecules that interfere with theinteraction between PD-L1 and PD-1 (e.g., a PD-L2-Fc fusion).

As used herein, a “PD-L1 binding antagonist” is a molecule thatdecreases, blocks, inhibits, abrogates or interferes with signaltransduction resulting from the interaction of PD-L1 with either one ormore of its binding partners, such as PD-1 and/or B7-1. In someembodiments, a PD-L1 binding antagonist is a molecule that inhibits thebinding of PD-L1 to its binding partners. In a specific aspect, thePD-L1 binding antagonist inhibits binding of PD-L1 to PD-1 and/or B7-1.In some embodiments, PD-L1 binding antagonists include anti-PD-L1antibodies and antigen-binding fragments thereof, immunoadhesins, fusionproteins, oligopeptides, small molecule antagonists, polynucleotideantagonists, and other molecules that decrease, block, inhibit, abrogateor interfere with signal transduction resulting from the interaction ofPD-L1 with one or more of its binding partners, such as PD-1 and/orB7-1. In one embodiment, a PD-L1 binding antagonist reduces the negativesignal mediated by or through cell surface proteins expressed on Tlymphocytes, and other cells, mediated signaling through PD-L1 or PD-1so as render a dysfunctional T-cell less dysfunctional. In someembodiments, a PD-L1 binding antagonist is an anti-PD-L1 antibody. In aspecific aspect, an anti-PD-L1 antibody is YW243.55.S70. In anotherspecific aspect, an anti-PD-L1 antibody is MDX-1105. In still anotherspecific aspect, an anti-PD-L1 antibody is atezolizumab (MPDL3280A). Instill another specific aspect, an anti-PD-L1 antibody is MED14736(druvalumab). In still another specific aspect, an anti-PD-L1 antibodyis MSB0010718C (avelumab).

As used herein, a “PD-1 binding antagonist” is a molecule thatdecreases, blocks, inhibits, abrogates or interferes with signaltransduction resulting from the interaction of PD-1 with one or more ofits binding partners, such as PD-L1 and/or PD-L2. In some embodiments,the PD-1 binding antagonist is a molecule that inhibits the binding ofPD-1 to its binding partners. In a specific aspect, the PD-1 bindingantagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. Forexample, PD-1 binding antagonists include anti-PD-1 antibodies andantigen-binding fragments thereof, immunoadhesins, fusion proteins,oligopeptides, small molecule antagonists, polynucleotide antagonists,and other molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-1 withPD-L1 and/or PD-L2. In one embodiment, a PD-1 binding antagonist reducesthe negative signal mediated by or through cell surface proteinsexpressed on T lymphocytes, and other cells, mediated signaling throughPD-1 or PD-L1 so as render a dysfunctional T-cell less dysfunctional. Insome embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody.In a specific aspect, a PD-1 binding antagonist is MDX-1106 (nivolumab).In another specific aspect, a PD-1 binding antagonist is MK-3475(pembrolizumab). In another specific aspect, a PD-1 binding antagonistis CT-011 (pidilizumab). In another specific aspect, a PD-1 bindingantagonist is MEDI-0680 (AMP-514). In another specific aspect, a PD-1binding antagonist is PDR001. In another specific aspect, a PD-1 bindingantagonist is REGN2810 described herein. In another specific aspect, aPD-1 binding antagonist is BGB-108 described herein. In another specificaspect, a PD-1 binding antagonist is AMP-224.

The term “vascular endothelial growth factor” or “VEGF” refers tovascular endothelial growth factor. The term “VEGF” encompasseshomologues and isoforms thereof. The term “VEGF” also encompasses theknown isoforms, e.g., splice isoforms, of VEGF, e.g., VEGF₁₁₁, VEGF₁₂₁,VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆, together with thenaturally-occurring allelic and processed forms thereof, including the110-amino acid human vascular endothelial cell growth factor generatedby plasmin cleavage of VEGF₁₆₅ as described in Ferrara Mol. Biol. Cell.21:687 (2010), Leung et al., Science, 246:1306 (1989), and Houck et al.,Mol. Endocrin., 5:1806 (1991). The term “VEGF” also refers to VEGFs fromnon-human species such as mouse, rat or primate. Sometimes the VEGF froma specific species are indicated by terms such as hVEGF for human VEGF,mVEGF for murine VEGF, and the like. The term “VEGF” is also used torefer to truncated forms of the polypeptide comprising amino acids 8 to109 or 1 to 109 of the 165-amino acid human vascular endothelial cellgrowth factor. Reference to any such forms of VEGF may be identified inthe present application, e.g., by “VEGF₁₀₉,” “VEGF (8-109),” “VEGF(1-109)” or “VEGF₁₆₅.” The amino acid positions for a “truncated” nativeVEGF are numbered as indicated in the native VEGF sequence. For example,amino acid position 17 (methionine) in truncated native VEGF is alsoposition 17 (methionine) in native VEGF. The truncated native VEGF hasbinding affinity for the KDR and Flt-1 receptors comparable to nativeVEGF. The term “VEGF variant” as used herein refers to a VEGFpolypeptide which includes one or more amino acid mutations in thenative VEGF sequence. Optionally, the one or more amino acid mutationsinclude amino acid substitution(s). For purposes of shorthanddesignation of VEGF variants described herein, it is noted that numbersrefer to the amino acid residue position along the amino acid sequenceof the putative native VEGF (provided in Leung et al., supra and Houcket al., supra).

The term “VEGF antagonist,” as used herein, refers to a molecule capableof binding to VEGF, reducing VEGF expression levels, or neutralizing,blocking, inhibiting, abrogating, reducing, or interfering with VEGFbiological activities, including, but not limited to, VEGF binding toone or more VEGF receptors, VEGF signaling, and VEGF-mediatedangiogenesis and endothelial cell survival or proliferation. Forexample, a molecule capable of neutralizing, blocking, inhibiting,abrogating, reducing, or interfering with VEGF biological activities canexert its effects by binding to one or more VEGF receptor (VEGFR) (e.g.,VEGFR1, VEGFR2, VEGFR3, membrane-bound VEGF receptor (mbVEGFR), orsoluble VEGF receptor (sVEGFR)). Included as VEGF antagonists useful inthe methods of the invention are polypeptides that specifically bind toVEGF, anti-VEGF antibodies and antigen-binding fragments thereof,receptor molecules and derivatives which bind specifically to VEGFthereby sequestering its binding to one or more receptors, fusionsproteins (e.g., VEGF-Trap (Regeneron)), and VEGF₁₂₁-gelonin (Peregrine).VEGF antagonists also include antagonist variants of VEGF polypeptides,antisense nucleobase oligomers complementary to at least a fragment of anucleic acid molecule encoding a VEGF polypeptide; small RNAscomplementary to at least a fragment of a nucleic acid molecule encodinga VEGF polypeptide; ribozymes that target VEGF; peptibodies to VEGF; andVEGF aptamers. VEGF antagonists also include polypeptides that bind toVEGFR, anti-VEGFR antibodies, and antigen-binding fragments thereof, andderivatives which bind to VEGFR thereby blocking, inhibiting,abrogating, reducing, or interfering with VEGF biological activities(e.g., VEGF signaling), or fusions proteins.

VEGF antagonists also include nonpeptide small molecules that bind toVEGF or VEGFR and are capable of blocking, inhibiting, abrogating,reducing, or interfering with VEGF biological activities. Thus, the term“VEGF activities” specifically includes VEGF-mediated biologicalactivities of VEGF. In certain embodiments, the VEGF antagonist reducesor inhibits, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% ormore, the expression level or biological activity of VEGF. In someembodiments, the VEGF inhibited by the VEGF-specific antagonist is VEGF(8-109), VEGF (1-109), or VEGF₁₆₅.

As used herein, VEGF antagonists can include, but are not limited to,anti-VEGFR2 antibodies and related molecules (e.g., ramucirumab,tanibirumab, aflibercept), anti-VEGFR1 antibodies and related molecules(e.g., icrucumab, aflibercept (VEGF Trap-Eye; EYLEA®), andziv-aflibercept (VEGF Trap; ZALTRAP®)), bispecific VEGF antibodies(e.g., MP-0250, vanucizumab (VEGF-ANG2), and bispecific antibodiesdisclosed in US 2001/0236388), bispecific antibodies includingcombinations of two of anti-VEGF, anti-VEGFR1, and anti-VEGFR2 arms,anti-VEGF antibodies (e.g., bevacizumab, sevacizumab, and ranibizumab),and nonpeptide small molecule VEGF antagonists (e.g., pazopanib,axitinib, vandetanib, stivarga, cabozantinib, lenvatinib, nintedanib,orantinib, telatinib, dovitinig, cediranib, motesanib, sulfatinib,apatinib, foretinib, famitinib, and tivozanib).

The terms “anti-VEGF antibody,” an “antibody that binds to VEGF,” and“antibody that specifically binds VEGF” refer to an antibody that iscapable of binding VEGF with sufficient affinity such that the antibodyis useful as a diagnostic and/or therapeutic agent in targeting VEGF. Inone embodiment, the extent of binding of an anti-VEGF antibody to anunrelated, non-VEGF protein is less than about 10% of the binding of theantibody to VEGF as measured, for example, by a radioimmunoassay (RIA).In certain embodiments, an antibody that binds to VEGF has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g., from 10⁻⁸ M to 10⁻¹³M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certain embodiments, an anti-VEGFantibody binds to an epitope of VEGF that is conserved among VEGF fromdifferent species.

In certain embodiments, the anti-VEGF antibody can be used as atherapeutic agent in targeting and interfering with diseases orconditions wherein the VEGF activity is involved. Also, the antibody maybe subjected to other biological activity assays, e.g., in order toevaluate its effectiveness as a therapeutic. Such assays are known inthe art and depend on the target antigen and intended use for theantibody. Examples include the HUVEC inhibition assay; tumor cell growthinhibition assays (as described in WO 89/06692, for example);antibody-dependent cellular cytotoxicity (ADCC) and complement-mediatedcytotoxicity (CDC) assays (U.S. Pat. No. 5,500,362); and agonisticactivity or hematopoiesis assays (see WO 95/27062). An anti-VEGFantibody will usually not bind to other VEGF homologues such as VEGF-Bor VEGF-C, nor other growth factors such as PIGF, PDGF, or bFGF. In oneembodiment, anti-VEGF antibody is a monoclonal antibody that binds tothe same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced byhybridoma ATCC HB 10709. In another embodiment, the anti-VEGF antibodyis a recombinant humanized anti-VEGF monoclonal antibody generatedaccording to Presta et al. (1997) Cancer Res. 57:4593-4599, includingbut not limited to the antibody known as bevacizumab (BV; AVASTIN®).

The anti-VEGF antibody “ranibizumab” also known as “LUCENTIS®” or“rhuFab V2” is a humanized, affinity-matured anti-human VEGF Fabfragment. Ranibizumab is produced by standard recombinant technologymethods in Escherichia coli expression vector and bacterialfermentation. Ranibizumab is not glycosylated and has a molecular massof ˜48,000 daltons. See WO 98/45331 and US 2003/0190317. Additionalpreferred antibodies include the G6 or B20 series antibodies (e.g.,G6-31, B20-4.1), as described in PCT Application Publication Nos. WO2005/012359 and WO 2005/044853, which are each incorporated herein byreference in their entirety. For additional preferred antibodies seeU.S. Pat. Nos. 7,060,269, 6,582,959, 6,703,020; 6,054,297; WO98/45332;WO 96/30046; WO94/10202; EP 0666868B1; U.S. Patent ApplicationPublication Nos. 2006009360, 20050186208, 20030206899, 20030190317,20030203409, and 20050112126; and Popkov et al., Journal ofImmunological Methods 288:149-164 (2004). Other preferred antibodiesinclude those that bind to a functional epitope on human VEGF comprisingof residues F17, M18, D19, Y21, Y25, Q89, 191, K101, E103, and C104 or,alternatively, comprising residues F17, Y21, Q22, Y25, D63, 183, andQ89. Additional anti-VEGF antibodies include anti-VEGF antibodiesdescribed in PCT Application Publication No. WO 2009/155724.

The term “co-administered” is used herein to refer to administration oftwo or more therapeutic agents, where at least part of theadministration overlaps in time. Accordingly, co-administration includesa dosing regimen when the administration of one or more agent(s)continues after discontinuing the administration of one or more otheragent(s).

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder,” and “tumor” are notmutually exclusive as referred to herein.

III. Methods

A. Diagnostic Methods

Provided herein are methods for diagnosing cancer (e.g., a lung cancer(e.g., squamous NSCLC or non-squamous NSCLC) or a head and neck cancer(e.g., HNSC)) in a subject. Also provided herein are methods foridentifying a subject having a cancer that is a NRF2-dependent cancer(e.g., lung cancer, e.g., squamous non-small cell lung cancer ornon-squamous non-small cell lung cancer, or head and neck cancer). Anyof the methods may be based on the expression level of a biomarkerprovided herein, for example, a splice variant of NRF2 (e.g., NRF2 mRNAor NRF2 protein), or an increased expression of one or more NRF2 targetgenes. Any of the methods may further include administering to thesubject a NRF2 pathway antagonist. Any of the methods may furtherinclude administering an effective amount of a second therapeutic (e.g.,one or more (e.g., 1, 2, 3, or 4 or more) additional NRF2 pathwayantagonists or one or more (e.g., 1, 2, 3, or 4 or more) anti-canceragents) to the subject.

The invention provides a method of diagnosing a cancer in a subject, themethod comprising determining the expression level of at least one gene(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, or 27 genes) selected from the groupconsisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1,KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15,NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in asample obtained from the subject; and comparing the expression level ofthe at least one gene to a reference expression level of the at leastone gene, wherein an increase in the expression level of the at leastone gene in the sample relative to the reference expression level of theat least one gene identifies a subject having a cancer.

The invention further provides a method of identifying a subject havinga cancer that is a NRF2-dependent cancer, the method comprisingdetermining the expression level of at least one gene (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, or 27 genes) selected from the group consisting of AKR1B10,AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11,TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1,GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sample obtained fromthe subject; comparing the expression level of the at least one gene toa reference expression level of the at least one gene; and determiningif the subject's cancer is a NRF2-dependent cancer, wherein an increasein the expression level of the at least one gene in the sample relativeto the reference expression level of the at least one gene identifies asubject having a NRF2-dependent cancer.

In any of the preceding methods, the expression level of one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or 21) of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16,KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, NR0B1, UGDH, TXNRD1, GSR, AKR1C3,TALDO1, PGD, TXN, or NQO1 is determined.

In any of the preceding methods, the expression level of one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) newly identified NRF2target genes is determined. Newly identified NRF2 target genes includeAKR1B10, AKR1C2, ME1, KYNU, CABYR, TRIM16L, AKR1C4, CYP4F11, RSPO3,AKR1B15, NR0B1, and AKR1C3.

The invention further provides a method of diagnosing a cancer in asubject, the method comprising determining the mRNA expression level ofNRF2 comprising a deletion in all or a portion of its exon 2 in a sampleobtained from the subject (e.g., a tumor sample), wherein the presenceof NRF2 comprising a deletion in all or a portion of its exon 2identifies the subject as having a cancer. In some embodiments, the NRF2further comprises a deletion in all or a portion of its exon 3. Presenceand/or expression levels of a gene (e.g., NRF2, KEAP1, AKR1B10, AKR1C2,SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L,AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR,AKR1C3, TALDO1, PGD, TXN, NQO1, or FTL) may be determined qualitativelyor quantitatively based on any suitable criterion known in the art,including, but not limited to DNA, mRNA, cDNA, protein fragments, and/orgene copy number.

The invention further provides a method of diagnosing a cancer in asubject, the method comprising determining the protein expression levelof NRF2 comprising a deletion in all or a portion of its Neh2 domain ina sample obtained from the subject, wherein the presence of NRF2comprising a deletion in all or a portion of its Neh2 domain identifiesthe subject as having a cancer. In some embodiments, the NRF2 furthercomprises a deletion in all or a portion of its Neh4 domain.

The invention further provides a method of identifying a subject havingcancer, the method comprising determining the mRNA expression level ofNRF2 comprising a deletion in all or a portion of its exon 2 in a sampleobtained from the subject (e.g., a tumor sample), wherein the presenceof NRF2 comprising a deletion in all or a portion of its exon 2identifies the subject as having a cancer. In some embodiments, the NRF2further comprises a deletion in all or a portion of its exon 3. Presenceand/or expression levels of a gene (e.g., NRF2, KEAP1, AKR1B10, AKR1C2,SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L,AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR,AKR1C3, TALDO1, PGD, TXN, NQO1, or FTL) may be determined qualitativelyor quantitatively based on any suitable criterion known in the art,including, but not limited to DNA, mRNA, cDNA, protein fragments, and/orgene copy number.

The invention further provides a method of identifying a subject havingcancer, the method comprising determining the protein expression levelof NRF2 comprising a deletion in all or a portion of its Neh2 domain ina sample obtained from the subject, wherein the presence of NRF2comprising a deletion in all or a portion of its Neh2 domain identifiesthe subject as having a cancer. In some embodiments, the NRF2 furthercomprises a deletion in all or a portion of its Neh4 domain.

The presence and/or expression level/amount of various biomarkersdescribed herein in a sample can be analyzed by a number ofmethodologies, many of which are known in the art and understood by theskilled artisan, including, but not limited to, immunohistochemistry(“IHC”), Western blot analysis, immunoprecipitation, molecular bindingassays, ELISA, ELIFA, fluorescence activated cell sorting (“FACS”),MassARRAY, proteomics, quantitative blood based assays (e.g., SerumELISA), biochemical enzymatic activity assays, in situ hybridization,fluorescence in situ hybridization (FISH), Southern analysis, Northernanalysis, whole genome sequencing, massively parallel DNA sequencing(e.g., next-generation sequencing), NANOSTRING®, polymerase chainreaction (PCR) including quantitative real time PCR (qRT-PCR) and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like, RNA-seq, microarray analysis, geneexpression profiling, and/or serial analysis of gene expression(“SAGE”), as well as any one of the wide variety of assays that can beperformed by protein, gene, and/or tissue array analysis. Typicalprotocols for evaluating the status of genes and gene products arefound, for example in Ausubel et al., eds., 1995, Current Protocols InMolecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting),15 (Immunoblotting) and 18 (PCR Analysis). Multiplexed immunoassays suchas those available from Rules Based Medicine or Meso Scale Discovery(“MSD”) may also be used.

In some embodiments of any of the methods described herein, DNA fromclinical tumor samples can be sequenced using a next-generationsequencing method, such as the targeted gene pulldown and sequencingmethod described in Frampton et al. (Nature Biotechnology. 31(11):1023-1033, 2013), which is incorporated by reference herein in itsentirety. Such a next-generation sequencing method can be used with anyof the methods disclosed herein to detect various mutations (e.g.,insertions, deletions, base substitutions, focal gene amplifications,and/or homozygous gene deletions), while enabling the use of smallsamples (e.g., from small-core needle biopsies, fine-needle aspirations,and/or cell blocks) or fixed samples (e.g., formalin-fixed andparaffin-embedded (FFPE) samples).

In any of the preceding methods, the presence and/or expressionlevel/amount of a biomarker (e.g., NRF2, KEAP1, AKR1B10, AKR1C2, SRXN1,OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4,CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3,TALDO1, PGD, TXN, NQO1, or FTL) is measured by determining proteinexpression levels of the biomarker. In certain embodiments, the methodcomprises contacting the biological sample with antibodies thatspecifically bind to a biomarker (e.g., anti-NRF2 antibodies) underconditions permissive for binding of the biomarker, and detectingwhether a complex is formed between the antibodies and biomarker. Suchmethod may be an in vitro or in vivo method. Any method of measuringprotein expression levels known in the art or provided herein may beused. For example, in some embodiments, a protein expression level of abiomarker is determined using a method selected from the groupconsisting of flow cytometry (e.g., fluorescence-activated cell sorting(FACS™)), Western blot, enzyme-linked immunosorbent assay (ELISA),immunoprecipitation, immunohistochemistry (IHC), immunofluorescence,radioimmunoassay, dot blotting, immunodetection methods, HPLC, surfaceplasmon resonance, optical spectroscopy, mass spectrometry, and HPLC. Insome embodiments, the protein expression level of the biomarker isdetermined in tumor cells.

In some embodiments, the presence and/or expression level/amount of abiomarker (e.g., NRF2, KEAP1, AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH,GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11,RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD,TXN, NQO1, or FTL) is measure by determining mRNA expression levels ofthe biomarker. In certain embodiments, presence and/or expressionlevel/amount of a gene is determined using a method comprising: (a)performing gene expression profiling, PCR (such as RT-PCR), RNA-seq,microarray analysis, SAGE, MassARRAY technique, or FISH on a sample(such as a subject cancer sample); and b) determining presence and/orexpression level/amount of a biomarker in the sample. In one embodiment,the PCR method is qRT-PCR. In one embodiment, the PCR method ismultiplex-PCR. In some embodiments, gene expression is measured bymicroarray. In some embodiments, gene expression is measured by qRT-PCR.In some embodiments, expression is measured by multiplex-PCR.

Methods for the evaluation of mRNAs in cells are well known and include,for example, hybridization assays using complementary DNA probes (suchas in situ hybridization using labeled riboprobes specific for the oneor more genes, Northern blot and related techniques) and various nucleicacid amplification assays (such as RT-PCR using complementary primersspecific for one or more of the genes, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like). Samples from mammals can be conveniently assayed for mRNAsusing Northern, dot blot, or PCR analysis. In addition, such methods caninclude one or more steps that allow one to determine the levels oftarget mRNA in a biological sample (e.g., by simultaneously examiningthe levels a comparative control mRNA sequence of a “housekeeping” genesuch as an actin family member).

In some embodiments of any of the methods, the biomarker is NRF2 (e.g.,exon 2-deleted NRF2 or exon 2+3-deleted NRF2). In one embodiment,expression level of biomarker is determined using a method comprisingperforming WGS analysis on a sample (such as a tumor sample obtainedfrom a patient) and determining expression level of a biomarker in thesample. In some embodiments, presence of exon 2-deleted NRF2 or exon2+3-deleted NRF2 is determined relative to a reference. In someembodiments, the reference is a reference value. In some embodiments,the reference is a reference sample (e.g., a control cell line sample, atissue sample from non-cancerous patient, or a wild-type NRF2 tissuesample).

Additionally or alternatively to mRNA expression analysis, otherbiomarkers, such as protein expression, may be quantified according tomethods described above. For example, methods of the invention includetesting a sample for a genomic biomarker (e.g., the presence of exon2-deleted NRF2 or exon 2+3-deleted NRF2, or the upregulation of one ormore NRF2 target genes, e.g., AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH,GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11,RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD,TXN, NQO1, or FTL) and additionally testing a sample for a proteinbiomarker (e.g., protein transcripts of one or more of AKR1B10, AKR1C2,SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L,AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR,AKR1C3, TALDO1, PGD, TXN, NQO1, or FTL).

In some embodiments of any of the methods, a DNA sequence may serve as abiomarker. DNA can be quantified according to any method known in theart, including, but not limited to, PCR, exome-seq (e.g., whole exomesequencing), DNA microarray analysis, NANOSTRING®, or whole genomesequencing.

In some instances, the expression level of the genes in the sample is anaverage (e.g., mean expression or median expression) of the genes, thereference expression level of the genes is an average (e.g., meanexpression or median expression) of the genes of the reference, and theaverage of the genes of the sample is compared to the average of thegenes of the reference.

In certain embodiments, the presence and/or expression levels/amount ofa biomarker in a first sample is increased or elevated as compared topresence/absence and/or expression levels/amount in a second sample. Incertain embodiments, the presence/absence and/or expressionlevels/amount of a biomarker in a first sample is decreased or reducedas compared to presence and/or expression levels/amount in a secondsample. In certain embodiments, the second sample is a reference sample,reference cell, reference tissue, control sample, control cell, orcontrol tissue. Additional disclosures for determining thepresence/absence and/or expression levels/amount of a gene are describedherein.

In certain embodiments, a reference sample, reference cell, referencetissue, control sample, control cell, or control tissue is a singlesample or combined multiple samples from the same subject or individualthat are obtained at one or more different time points than when thetest sample is obtained. For example, a reference sample, referencecell, reference tissue, control sample, control cell, or control tissueis obtained at an earlier time point from the same subject or individualthan when the test sample is obtained. Such reference sample, referencecell, reference tissue, control sample, control cell, or control tissuemay be useful if the reference sample is obtained during initialdiagnosis of cancer and the test sample is later obtained when thecancer becomes metastatic.

In certain embodiments, a reference sample, reference cell, referencetissue, control sample, control cell, or control tissue is a combinedmultiple samples from one or more healthy individuals who are not thepatient. In certain embodiments, a reference sample, reference cell,reference tissue, control sample, control cell, or control tissue is acombined multiple samples from one or more individuals with a disease ordisorder (e.g., cancer) who are not the subject or individual. Incertain embodiments, a reference sample, reference cell, referencetissue, control sample, control cell, or control tissue is pooled RNAsamples from normal tissues or pooled plasma or serum samples from oneor more individuals who are not the patient. In certain embodiments, areference sample, reference cell, reference tissue, control sample,control cell, or control tissue is pooled RNA samples from tumor tissuesor pooled plasma or serum samples from one or more individuals with adisease or disorder (e.g., cancer) who are not the patient.

In some embodiments of any of the methods, elevated or increasedexpression refers to an overall increase of about any of 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in thelevel of biomarker (e.g., protein or nucleic acid (e.g., gene (DNA ormRNA))), detected by standard art-known methods such as those describedherein, as compared to a reference sample, reference cell, referencetissue, control sample, control cell, or control tissue. In certainembodiments, the elevated expression refers to the increase inexpression level/amount of a biomarker in the sample wherein theincrease is at least about any of 1.5×, 1.75×, 2×, 3×, 4×, 5×, 6×, 7×,8×, 9×, 1 Ox, 25×, 50×, 75×, or 100× the expression level/amount of therespective biomarker in a reference sample, reference cell, referencetissue, control sample, control cell, or control tissue. In someembodiments, elevated expression refers to an overall increase ofgreater than about 1.5 fold, about 1.75 fold, about 2 fold, about 2.25fold, about 2.5 fold, about 2.75 fold, about 3.0 fold, or about 3.25fold as compared to a reference sample, reference cell, referencetissue, control sample, control cell, control tissue, or internalcontrol (e.g., housekeeping gene).

In some embodiments of any of the methods, reduced expression refers toan overall reduction of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of biomarker(e.g., protein or nucleic acid (e.g., gene (DNA or mRNA))), detected bystandard art known methods such as those described herein, as comparedto a reference sample, reference cell, reference tissue, control sample,control cell, or control tissue. In certain embodiments, reducedexpression refers to the decrease in expression level/amount of abiomarker in the sample wherein the decrease is at least about any of0.9×, 0.8×, 0.7×, 0.6×, 0.5×, 0.4×, 0.3×, 0.2×, 0.1×, 0.05×, or 0.01×the expression level/amount of the respective biomarker in a referencesample, reference cell, reference tissue, control sample, control cell,or control tissue.

B. Therapeutic Methods

The present invention provides methods for treating a patient sufferingfrom a cancer (e.g., a lung cancer (e.g., squamous NSCLC or non-squamousNSCLC) or a head and neck cancer (e.g., HNSC)). In some instances, themethods of the invention include administering to the patient aneffective amount of a NRF2 pathway antagonist. Any of the NRF2 pathwayantagonists described herein or otherwise known in the art may be usedin the methods. In some instances, the methods involve determining thepresence and/or expression level of a NRF2 splice variant (e.g., exon2-deleted NRF2 or exon 2+3-deleted NRF2) or a NRF2 target gene in asample obtained from a patient and administering an NRF2 pathwayantagonist to the patient based on the presence and/or expression levelof a NRF2 splice variant (e.g., exon 2-deleted NRF2 or exon 2+3-deletedNRF2) or a NRF2 target gene, e.g., using any of the methods describedherein, in the Examples below, or known in the art.

The invention provides a method of treating a subject suffering from acancer (e.g., a lung cancer (e.g., squamous NSCLC or non-squamous NSCLC)or a head and neck cancer (e.g., HNSC)), the method comprisingdetermining the expression level of at least one gene (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, or 27 genes) selected from the group consisting of AKR1B10,AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11,TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1,GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sample obtained fromthe subject; and comparing the expression level of the at least one geneto a reference expression level of the at least one gene, wherein anincrease in the expression level of the at least one gene in the samplerelative to the reference expression level of the at least one geneidentifies a subject having a cancer, and administering to the subject atherapeutically effective amount of one or more NRF2 pathwayantagonists.

The invention further provides a method of treating a subject sufferingfrom a cancer (e.g., lung cancer (e.g., squamous NSCLC or non-squamousNSCLC) or head and neck cancer), wherein the expression level of one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) newly identifiedNRF2 target genes is determined. Newly identified NRF2 target genesinclude AKR1B10, AKR1C2, ME1, KYNU, CABYR, TRIM16L, AKR1C4, CYP4F11,RSPO3, AKR1B15, NR0B1, and AKR1C3.

In some instances, the invention further provides a method of treating asubject suffering from a cancer (e.g., lung cancer (e.g., squamous NSCLCor non-squamous NSCLC) or head and neck cancer), wherein the mRNAexpression level of NRF2 comprises a deletion in all, or a portion of,its exon 2 in a sample obtained from the subject, and wherein thepresence of NRF2 comprising a deletion in all or a portion of its exon 2identifies the subject as having a cancer; and administering to thesubject a therapeutically effective amount of one or more NRF2 pathwayantagonists. In some embodiments, the NRF2 further comprises a deletionin all, or a portion of, its exon 3.

In some instances, the invention further provides a method of treating asubject suffering from a cancer (e.g., a lung cancer (e.g., squamousNSCLC or non-squamous NSCLC) or a head and neck cancer (e.g., HNSC)),wherein the NRF2 protein comprises a deletion in all, or a portion of,its Neh2 domain in a sample obtained from the subject, and wherein thepresence of NRF2 comprising a deletion in all, or a portion of, its Neh2domain identifies the subject as having a cancer; and administering tothe subject a therapeutically effective amount of one or more NRF2pathway antagonists. In some embodiments, the NRF2 further comprises adeletion in all or a portion of its Neh4 domain.

In any of the preceding methods, the NRF2 pathway antagonist may be anyNRF2 pathway antagonist known in the art or described herein.

In some instances, the method further includes administering to thesubject an effective amount of a second therapeutic agent (e.g., one ormore anti-cancer agents). In some instances, the second therapeuticagent is selected from the group consisting of an anti-angiogenic agent,a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent,an immunotherapy, and combinations thereof. In some embodiments, theimmunotherapy is a VEGF antagonist (e.g., anti-VEGFR2 antibodies andrelated molecules (e.g., ramucirumab, tanibirumab, aflibercept),anti-VEGFR1 antibodies and related molecules (e.g., icrucumab,aflibercept (VEGF Trap-Eye; EYLEA®), and ziv-aflibercept (VEGF Trap;ZALTRAP®)), bispecific VEGF antibodies (e.g., MP-0250, vanucizumab(VEGF-ANG2), and bispecific antibodies disclosed in US 2001/0236388),bispecific antibodies including combinations of two of anti-VEGF,anti-VEGFR1, and anti-VEGFR2 arms, anti-VEGF antibodies (e.g.,bevacizumab, sevacizumab, and ranibizumab), and nonpeptide smallmolecule VEGF antagonists (e.g., pazopanib, axitinib, vandetanib,stivarga, cabozantinib, lenvatinib, nintedanib, orantinib, telatinib,dovitinig, cediranib, motesanib, sulfatinib, apatinib, foretinib,famitinib, and tivozanib)). In other embodiments, the immunotherapy is aPD-1 axis binding antagonist (e.g., YW243.55.S70, MDX-1105, MPDL3280A(atezolizumab), MEDI4736 (druvalumab), MSB0010718C (avelumab), MDX-1106(nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680(AMP-514), PDR001, REGN2810, BGB-108 or AMP-224).

The compositions used in the methods described herein (e.g., NRF2pathway antagonists) can be administered by any suitable method,including, for example, intravenously, intramuscularly, subcutaneously,intradermally, percutaneously, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intrathecally, intranasally,intravaginally, intrarectally, topically, intratumorally, peritoneally,subconjunctivally, intravesicularly, mucosally, intrapericardially,intraumbilically, intraocularly, intraorbitally, orally, topically,transdermally, intravitreally (e.g., by intravitreal injection), by eyedrop, by inhalation, by injection, by implantation, by infusion, bycontinuous infusion, by localized perfusion bathing target cellsdirectly, by catheter, by lavage, in cremes, or in lipid compositions.The compositions utilized in the methods described herein can also beadministered systemically or locally. The method of administration canvary depending on various factors (e.g., the compound or compositionbeing administered and the severity of the condition, disease, ordisorder being treated). In some embodiments, the NRF2 pathwayantagonist is administered intravenously, intramuscularly,subcutaneously, topically, orally, transdermally, intraperitoneally,intraorbitally, by implantation, by inhalation, intrathecally,intraventricularly, or intranasally. Dosing can be by any suitableroute, e.g., by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic. Various dosing schedules including but not limited to single ormultiple administrations over various time-points, bolus administration,and pulse infusion are contemplated herein.

NRF2 pathway antagonists described herein (and any additionalanti-cancer agents) may be formulated, dosed, and administered in afashion consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. TheNRF2 pathway antagonist need not be, but is optionally formulated withand/or administered concurrently with one or more agents currently usedto prevent or treat the disorder in question. The effective amount ofsuch other agents depends on the amount of the Nrd2 pathway inhibitorpresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

In some embodiments, the methods further involve administering to thepatient an effective amount of a second therapeutic agent (e.g., one ormore anti-cancer agents). In some embodiments, the second therapeuticagent is selected from the group consisting of an anti-angiogenic agent,a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent,an immunotherapy, and combinations thereof.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents (e.g., a NRF2 pathway antagonistand an anti-cancer agent) are included in the same or separateformulations), and separate administration, in which case,administration of a NRF2 pathway antagonist can occur prior to,simultaneously, and/or following, administration of the additionalanti-cancer agent or agents. In one embodiment, administration of NRF2pathway antagonist and administration of an additional anti-cancer agentoccur within about one month, or within about one, two or three weeks,or within about one, two, three, four, five, or six days, of each other.

C. NRF2 Pathway Antagonists for Use in the Methods of the Invention

Provided herein are methods for treating or delaying progression of acancer (e.g., a lung cancer (e.g., squamous NSCLC) or head and neckcancer) in a subject comprising administering to the subject atherapeutically effective amount of a NRF2 pathway antagonist. Any ofthe preceding methods may be based on the expression level of abiomarker provided herein, for example, NRF2 expression or expression ofany protein or mRNA involved in a NRF2 pathway in a tumor sample, e.g.,a biopsy containing tumor cells.

In some embodiments, a NRF2 pathway antagonist is a small molecule,e.g., a small molecule capable of binding to NRF2 or protein or genethat regulates the expression, stability, or activity of NRF2.

In some embodiments, the NRF2 pathway antagonist is an antagonist of aNRF2 agonist. Examples of NRF2 agonists include, but are not limited to,cAMP response element-binding protein (CREB), CREB Binding Protein(CBP), Maf, activating transcription factor 4 (ATF4), protein kinase C(PKC), Jun, glucocorticoid receptor, UbcM2, and homologous to the E6-APcarboxyl terminus domain and Ankyrin repeat containing E3 ubiquitinprotein ligase 1 (HACE1). Therefore, examples of NRF2 pathwayantagonists include, but are not limited to, CREB antagonists, CBPantagonists, Maf antagonists, ATF4 antagonists, PKC antagonists, Junantagonists, glucocorticoid receptor antagonists, UbcM2 antagonists, andHACE1 antagonists, such as those set forth in Table 2.

In some embodiments, the NRF2 pathway antagonist is an agonist of a NRF2antagonist. Examples of NRF2 antagonists include, but are not limitedto, c-Myc, SUMO, KEAP1, CUL3, retinoic acid receptor α (RARα).Therefore, examples of NRF2 pathway antagonists include, but are notlimited to, c-Myc agonists, SUMO, KEAP1 agonists, CUL3 agonists, andRARα agonists, such as those set forth in Table 3.

TABLE 2 Compound Target KG-501 CREB 2-naphthol-AS-E-phosphate C646 CBP4-[4-[[5-(4,5-Dimethyl-2-nitrophenyl)-2-furanyl]methylene]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzoic acid CBP30 CBP8-(3-chloro-4-methoxy-phenethyl)-4-(3,5-dimethyl-isoxazol-4-yl)-9-(2-(morpholin-4-yl)-propyl)-7,9-diaza-bicyclo[4.3.0]nona-1(6),2,4,7-tetraene nivalenolc-maf 3,4,7,15-Tetrahydroxy-12,13-epoxytrichothec-9-en-8-on tomatidineATF4 (3β,5α,22β,25S)-spirosolan-3-ol ruboxistaurin PKC(9S)-9-[(dimethylamino)methyl]-6,7,10,11-tetrahydro-9H,18H-5,21:12,17-di(metheno)dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecine-18,20-dioneSP600125 Jun 1,9-Pyrazoloanthrone mifepristone Glucocorticoid(11β,17β)-11-[4-(Dimethylamino)phenyl]-17-hydroxy-17-(1-propynyl)-estra-4,9-dien-receptor 3-one CORT 108297 Glucocorticoid1H-Pyrazolo[3,4-g]isoquinoline,4a-(ethoxymethyl)-1-(4-fluorophenyl)-4,4a,5,6,7,8- receptorhexahydro-6[[4-(trifluoromethyl)phenyl]sulfonyl]-, (4aR)-

TABLE 3 Compound Target Al-1 KEAP14-Chloro-1,2-dihydro-1-methyl-2-oxo-3-quinolinecarboxylic acid ethylester, Ethyl 4- chloro-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylateretinoic acid RARα3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2E,4E,6E,8E,-nonatetraenoicacid CD437 RARα6-(4-Hydroxy-3-tricyclo[3.3.1.13,7]dec-1-ylphenyl)-2-naphthalenecarboxylicacid TTNPB RARα4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoicacid

In some embodiments of the invention, derivatives of the compoundslisted in Table 2 or 3 may also be administered as NRF2 pathwayantagonists. A derivative of a compound listed in Table 2 or 3 is asmall molecule that differs in structure from the parent compound, butretains the ability to antagonize a NRF2 pathway. A derivative of acompound may change its interaction with certain other molecules orproteins relative to the parent compound. A derivative of a compound mayalso include a salt, an adduct, or other variant of the parent compound.In some embodiments of the invention, any derivative of a compounddescribed herein (e.g., any one compound of the compounds listed inTable 2 or 3 may be used instead of the parent compound. In someembodiments, any derivative of a compound listed in Table 2 or 3 may beused in a method of treating a subject having cancer, such as lungcancer.

In some embodiments, a NRF2 pathway antagonist is an antibody (e.g., ananti-NRF2 antibody or an antibody directed against a protein or genethat regulates NRF2 expression, stability, or activity, e.g., a targetlisted in Table 2 or 3). In some embodiments, the anti-NRF2 antibody iscapable of inhibiting binding between NRF2 and antioxidant responseelement. In some embodiments, the anti-NRF2 antibody is capable ofinhibiting binding between NRF2 and a cofactor (e.g., Maf, PKC, Jun,ATF4, or CBP). In some embodiments, the antibody of the invention is anantibody fragment selected from the group consisting of Fab, Fab′-SH,Fv, scFv, and (Fab′)₂ fragments. In some embodiments, the antibody is ahumanized antibody. In some embodiments, the antibody is a humanantibody. In some embodiments, the antibody is a derivative of a knownantibody having any of the above-mentioned properties. Derivatives ofantibodies include antibody variants having about 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% or lower sequence identity to itsparent antibody. Percent (%) amino acid sequence identity is determinedaccording to methods known in the art, including by ALIGN-2, asdescribed above.

In some embodiments, a NRF2 pathway antagonist includes an inhibitor ofany downstream biomarker (e.g., gene or protein, e.g., a gene or proteininvolved in iron sequestration (e.g., Ferritin, Light Polypeptide (FTL),Ferritin, Heavy Polypeptide 1 (FTH), or Heme Oxygenase 1 (HMOX1)), GSHutilization (e.g., Glutathione Peroxidase 2 (GPX2), GlutathioneS-Transferase Alpha 1 (GSTA1), Glutathione S-Transferase Alpha 2(GSTA2), Glutathione S-Transferase Alpha 3 (GSTA3), GlutathioneS-Transferase Alpha 5 (GSTA5), Glutathione S-Transferase Mu 1 (GSTM1),Glutathione S-Transferase Mu 2 (GSTM2), Glutathione S-Transferase Mu 3(GSTM3), or Glutathione S-Transferase Pi 1 (GSTP1)), quininedetoxification (e.g., NAD(P)H Dehydrogenase, Quinone 1 (NQO1)), GSHproduction and regeneration (e.g., Glutamate-Cysteine Ligase, ModifierSubunit (GCLM), Glutamate-Cysteine Ligase, Catalytic Subunit (GCLC),Glutathione Reductase (GSR), or Solute Carrier Family 7 (Anionic AminoAcid Transporter Light Chain, Xc-System), Member 11 (SLC7A11, or XCT)),thioredoxin (TXN) production, regeneration, and utilization (e.g.,Thioredoxin 1, (TXN1), Thioredoxin Reductase 1 (TXNRD1), orPeroxiredoxin 1 (PRDX1)), NADPH production (e.g., Glucose-6-PhosphateDehydrogenase (G6PD), Phosphogluconate Dehydrogenase (PGD), Malic Enzyme1, NADP(+)-Dependent, Cytosolic (ME1), Isocitrate Dehydrogenase 1(NADP+), Soluble (IDH1)) or any of the genes or proteins thereof ofTable 1).

In some embodiments, a NRF2 pathway antagonist includes a compound thatinhibits NRF2 from binding to antioxidant response element (ARE) (e.g.,by competitively binding to the ARE binding site on NRF2, bycompetitively binding to ARE, or by otherwise interfering with atranscriptional cofactor (e.g., small Maf proteins).

In some embodiments, a NRF2 pathway antagonist includes an agonist orantagonist of NRF2-related genes, such that the pharmacological effectof compound involves the downregulation of one or more pathwaysdownstream of NRF2-mediated transcription. Such NRF2-related genesinclude, e.g., Kelch-Like ECH-Associated Protein 1 (KEAP1),Ectodermal-Neural Cortex 1 (With BTB Domain) (ENC1), Protein Kinase C,Delta (PRKCD), Protein Kinase C, Beta (PRKCB), Polyamine-ModulatedFactor 1 (PMF1), Cullin 3 (CUL3), Nuclear Factor, Erythroid 2 (NFE2),Activating Transcription Factor 4 (ATF4), Heme Oxygenase 1 (HMOX1), HemeOxygenase 2 (HMOX2), Ubiquitin C (UBC), V-Maf Avian MusculoaponeuroticFibrosarcoma Oncogene Homolog K (MAFK), UDP Glucuronosyltransferase 1Family, Polypeptide A6 (UGT1A6), V-Maf Avian MusculoaponeuroticFibrosarcoma Oncogene Homolog F (MAFF), CREB Binding Protein (CREBBP),V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homolog G (MAFG),CAMP Responsive Element Binding Protein 1 (CREB1), FXYD DomainContaining Ion Transport Regulator 2 (FXYD2), Jun Proto-Oncogene (JUN),Small Ubiquitin-Like Modifier 2 (SUMO2), Small Ubiquitin-Like Modifier 1(SUMO1), V-Myc Avian Myelocytomatosis Viral Oncogene Homolog (MYC),Crystallin, Zeta (Quinone Reductase) (CRYZ), Aldo-Keto Reductase Family7, Member A2 (Aflatoxin Aldehyde Reductase) (AKR7A2), and GlutathioneS-Transferase Alpha 2 (GSTA2).

In some embodiments, a method of increasing ubiquitination of NRF2 in acell is provided, the method comprising contacting the cell with aninhibitor of a NRF2 pathway under conditions allowing inhibition of aNRF2 pathway in a cell. Increased ubiquitination of NRF2 can bedetermined, e.g., by immunoaffinity enrichment of ubiquitinated NRF2following trypsin digestion, followed by mass spectrometry, according toknown methods. In some embodiments, an increase in ubiquitation may bedetermined by comparing the ubiquitination of a wild-type NRF2 in a cellor population of cells contacted with a NRF2 pathway antagonist with theubiquitination of an exon 2 or exon 2+3 deleted NRF2 in a cell or apopulation of cells contacted with a NRF2 pathway antagonist and/or theubiquitination of an exon 2 or exon 2+3 deleted NRF2 in a cell or apopulation of cells not contacted with a NRF2 pathway antagonist.

In some embodiments of the invention, the NRF2 pathway antagonist isascorbic acid, brusatol, luteolin, or ochratoxin A.

Examples Example 1: Materials and Experimental Methods

A. Mutation and Copy Number Analysis

For 99 NSCLC cell lines, non-synonymous mutations and copy number datafor KRas, LKB1, KEAP1, and NRF2 were obtained from Klijn et al. (NatBiotechnol. 33(3):306-312, 2015). Thirteen additional NSCLC cell lineswere subjected to copy number analysis. In addition, exome sequencingwas applied to 104 NSCLC cell lines. For the cancer genome atlas (TCGA)tumors mutation and copy number data were retrieved from cBioPortalusing the R software package CGDS-R (Cerami et al. Cancer Discovery.2:401-404, 2012; Gao et al. Sci. Signal. 6:11, 2013).

B. RNA-Seq Analysis and Derivation of a Mutant KEAP1 Gene ExpressionSignature

Raw RNA-seq data for 99 NSCLC cell lines were retrieved from theEuropean Genome-phenome Archive (accession number EGAS00001000610)(PMID: 25485619). Mutations in KEAP1 and NRF2 in each of the NSCLC celllines are provided in Table 4. Raw RNA-seq data were downloaded fromTCGA and aligned to the human reference genome (GRCh37/hgl9) using GSNAPversion 2013-10-10 (Wu and Nacu. Bioinformatics 26:873-881, 2010),allowing maximum of 2 mismatches (parameters: “-M 2 -n 10 -B 2 -i 1 -N 1-w 200000 -E 1—pairmax-rna=200000”). Gene expression levels werequantified with RPKM (reads per kilobase of target and million readssequenced) values derived from the number of reads mapped to each RefSeqgene. Using the DESeq R package (PMID: 20979621) differential geneexpression was measured between KEAP1 mutant and KEAP1 wild-type celllines, reported as fold-change and associated adjusted p-values. Forward clustering of samples and genes (using Euclidean distance) invariance stabilized count data were used. The ‘NMF’ R package was usedto create associated heatmaps.

TABLE 4 Name Sample Type Gene Mutation BEN Carcinoma KEAP1 A556TNCI-H460 Carcinoma Large Cell KEAP1 D236H NCI-H838 Carcinoma Non-SmallCell KEAP1 E444* HCC44 Carcinoma Non-Small Cell KEAP1 F211C A549Carcinoma KEAP1 G333C HCC-15 Carcinoma Non-Small Cell KEAP1 G364CNCI-H1648 Adenocarcinoma KEAP1 G364C NCI-H2110 Carcinoma Non-Small CellKEAP1 G429C LXF-289 Adenocarcinoma KEAP1 G430V NCI-H647 CarcinomaNon-Small Cell KEAP1 G523W NCI-H920 Carcinoma Non-Small Cell KEAP1 G603VHCC4019 Adenocarcinoma KEAP1 K131* NCI-H23 Carcinoma Non-Small CellKEAP1 Q193H NCI-H1355 Adenocarcinoma KEAP1 Q75* NCI-H1915 CarcinomaNon-Small Cell KEAP1 R135L NCI-H2126 Carcinoma Non-Small Cell KEAP1R272C NCI-H1944 Carcinoma Non-Small Cell KEAP1 R272L NCI-H1623 CarcinomaNon-Small Cell KEAP1 R320L NCI-H2170 Carcinoma Squamous Cell KEAP1 R336*NCI-H1435 Carcinoma Non-Small Cell KEAP1 R413L NCI-H322T Unknown KEAP1R4605 H322T Carcinoma Non-Small Cell KEAP1 R4605 NCI-H661 CarcinomaLarge Cell KEAP1 V168I NCI-H2030 Carcinoma Non-Small Cell KEAP1 V568FNCI-H2023 Carcinoma Non-Small Cell KEAP1 W252C H1573 AdenocarcinomaKEAP1 A143P NCI-H2172 Carcinoma Non-Small Cell KEAP1 G430C H1792Adenocarcinoma KEAP1 G462W NCI-H2122 Carcinoma Non-Small Cell KEAP1R202G HCC2270 Adenocarcinoma NRF2 G31E NCI-H2228 Carcinoma Non-SmallCell NRF2 G31A NCI-H1568 Carcinoma Non-Small Cell NRF2 DEE77-79 EBC-1Carcinoma Non-Small Cell NRF2 D77V

C. Splice Variant Analysis

Analysis of splice variants was performed using the SGSeq softwarepackage available from the Bioconductor project website (Gentleman etal. Genome Biol. 5:R80, 2004). Exons and splice junctions were predictedfrom BAM files for 7,384 TCGA samples at 54 genomic loci of knownoncogenes using parameters alpha=2, psi=0, beta=0.2, gamma=0.2.Predicted features were merged across samples, and exons were processedinto disjoint exon bins. Splice junctions and exon bins were assembledinto a genome-wide splice graph. Splice events, which consist of two ormore alternative splice variants, were identified from the graph. Splicevariants were quantified in terms of FPKM and relative usage ψ. Briefly,local estimates of relative usage at the start and end of the variantwere obtained as the fraction of fragments that are compatible with thevariant. Estimates at the event start and end were combined using aweighted mean, with weights proportional to the total number offragments spanning the boundary. Relative usage estimates withdenominator less than 20 were set to NA. To obtain a local estimate ofabsolute expression at the variant start and end, compatible counts nwere converted to FPKMs as n I (N×L)×109 where N is the total number ofaligned fragments and L is the effective length (the number of allowedpositions for a compatible fragment). Splice variants detected in TCGAsamples were also quantified in 2,958 genotype-tissue expression project(GTEx) samples from normal human tissues (Consortium. Science.348:648-660, 2015).

D. Identification of Cancer-Specific Splice Variants

Only internal splice variants (not involving alternative transcriptstarts or ends) were considered and the start and end of each splicevariant were required to either overlap or extend exons that belong toannotated ref Gene transcripts downloaded from the UCSC Genome Browserwebsite (Pruitt et al. Nucleic Acids Res. 33:D501-504, 2005; Rosenbloomet al. Nucleic Acids Res. 43:D670-681, 2015). Retained introns wereexcluded. 19 TCGA indications that included at least 100 cancer samples(6,359 cancer samples in total) were considered and splice variants with(i) FPKM>2 and relative usage ψ>0.2 in at least one cancer sample and(ii) FPKM<1 in>99.9% of GTEx samples, and (iii) FPKM˜0 in>97.5% ofGTE×samples were selected. FPKM-based criteria were required to besatisfied at both the start and end of the splice variant. Variantssatisfying the FPKM-based criteria for which 4 could not be estimatedwere included after manual inspection.

E. Analysis of Targeted Paired-End Exome-Seq Data

All samples within FoundationCORE were processed and sequenced similarlyas previously described (Frampton et al. Nat. Biotechnol. 31, 1023-1031,2014). NRF2 exon 2 and exon 2+3 deletions were screened across aFoundationCORE dataset (n=58,707) using two distinct approaches.

First, rearrangement calls based on discordant read pairs and/or splitreads were examined for direct evidence for loss of NRF2 exon 2 or exon2+3. Although this approach provides direct evidence of the deletions ofinterest, deletions can only be discovered with this approach if thebreakpoints are within a baited region because intronic regions of NRF2are not captured. Thus, this approach identifies a limited subset ofNRF2 exon 2 or exon 2+3 deletions in which the breakpoints occur nearintron-exon boundaries or within exons.

The second approach utilizes copy number log ratio data from individualbait regions. Copy number log ratio values were determined with anin-house algorithm, educated to the specific tumor cellularity of eachsample. A z-score was calculated comparing the log ratio for each exonin NRF2 to control polymorphism capture regions immediately adjacent toNRF2 (n=15; evenly spaced every ˜1 MB from ˜3 MB upstream and ˜12 MBdownstream of NRF2). Exon 2 deletions with and without concurrent exon 3deletion were specifically examined. These are herein referred to asexons of interest (EOI). EOI deletions were called if (1) a z-score was<−2 for EOI and not for non-EOIs in NRF2 and (2) a log-ratio drop of 0.2from non-EOIs in NRF2 was calculated. Mutual exclusivity between NRF2exon 2 or exon 2+3 deletions and short variants in NRF2 or KEAP1 wasexamined specifically within lung squamous cell carcinoma (n=1,218).

F. Cell Culture

KMS-27 (RPMI-1640), JHH-6 (Williams Media E), HuCCT1 (RPMI-1640), andHUH-1 (DMEM) cells were from JCRB, and 293 (EMEM) cells were from ATCC.Cells were cultured in the indicated media in the presence of 2 mMglutamine and 10% FBS.

G. Western Blotting

Cell lysates were prepared with RIPA Buffer (Sigma) supplemented withcomplete EDTA-free protease inhibitor (Roche) and phoSTOP (Roche),Phosphatase Inhibitor Cocktail 2 (Sigma) and Phosphatase InhibitorCocktail 3 (Sigma) phosphatase inhibitors. Lysates were run on NovexTris-Glycine 4-12% gradient gels (ThermoFisher) and transferred ontoiBlot nitrocellulose (Invitrogen). Blots were pre-incubated in 5% skimmilk powder (Merck) in TBST (10 mM Tris pH8, 150 mM NaCl, 0.1%TWEEN-20), followed by 5% bovine serum albumin (Sigma) in TBSTcontaining antibodies. Secondary antibodies used were ECL Anti-RabbitHRP and ECL Anti-Mouse HRP (both from GE Heathcare). Blots weredeveloped with a Chemiluminescence Substrate Kit (Protein Simple) andvisualized with a FluorChem HD2 imager (Protein Simple). Antibodies usedin this study are against KEAP1 (Cell Signaling G1010), NRF2 (Abcamab62352), HSP90 (Cell Signaling 4877), HDAC2 (Cell Signaling 5113),β-actin (Sigma A2228), HA (Roche 11815016001), and FLAG (Sigma F2426).Lamda phosphatase was from NEB (P0753L), and phosphatase inhibitors wereomitted from the lysis buffer in these experiments.

H. Cell Viability and DNA Fragmentation Analysis

siRNAs were reverse transfected into cells with Dharmafect 2 reagent(ThermoFisher) and OptiMEM (Gibco). Four days post transfection, cellswere measured for viability using CellTiter-Glo reagent (Promega) andluminescence was detected on an EnVision Multi-label Reader (PerkinElmer). siRNAs were reverse transfected into cells with Dharmafect 2reagent (ThermoFisher) and OptiMEM (Gibco). Four days post-tranfection,cells were measured for apoptosis using propidium iodide (PI)(LifeTechnologies) staining and flow cytometry following a publishedprotocol (Riccardi and Nicoletti Nat. Protoc. 1:1458-1461, 2006).Staurosporin, 1 μM, (Enzo) was added to positive control cells 24 hourspre-staining. siRNAs targeting NRF2 exon 2 had the sequences:5′-TGGAGTAAGTCGAGAAGTA-3′ (SEQ ID NO: 29) and 5′-ACAACTAGATGAAGAGACA-3′(SEQ ID NO: 30). siRNAs targeting NRF2 exon 5 had the sequences:5′-TGACAGAAGTTGACAATTA-3′ (SEQ ID NO: 31) and 5′-GTAAGAAGCCAGATGTTAA-3′(SEQ ID NO: 32), and were used along with non-target siRNA as controlsiRNA. Stained cells were analyzed with a Becton Dickinson FACS Caliberinstrument. siRNAs targeting KEAP1 were from Dhamacon (L012453-00).

DNA fragmentation was quantified by propidium iodide (PI) staining andmeasured by flow cytometry according to Riccardi et al. (NatureProtocols, 1:1458-1461 (2006)).

I. Taqman Analysis

Total cellular RNA was extracted with an RNeasy Kit (Qiagen). RNA wasconverted to cDNA using a High Capacity cDNA Reverse Transcription Kit(Applied Biosystems), and cDNAs were amplified with Taqman GeneExpression primer-probe sets (ThermoFisher) using Taqman Gene ExpressionMaster Mix reagents (Applied Biosystems). Taqman amplification/detectionwas performed on a QuantStudio7 Flex Real-Time PCR System. Primer-probesets used were Hs00232352_ml and Hs00975961_gl to detect NRF2 exons 2and 5, respectively (ThermoFisher). NRF2 target gene Taqman primer-probesets used were: SLC7A11 (Hs00921938_ml), SGRN (Hs00921938_ml), NR0B1(Hs03043658_ml), GCLC (HsOOl55249_ml), and GPX2 (Hs01591589_ml), allfrom ThermoFisher.

J. 293 Transfections

Plasmid DNAs were transfected into cells using Lipofectamine 2000(ThermoFisher) and OptiMEM (Gibco) as recommended by manufacturerprotocol. Lysates were prepared 2-3 days post transfection. Expressionplasmids used were pRK5.NRF2, pRK5.NRF2.delta.e2, pRK5.NRF2.delta.e2,3,pRK5.NRF2.FLAG and pRK5.KEAP1.HA.

K. Tumor Xenograft Models

Eleven to twelve week-old female C.B-17 SCID.beige mice (Charles RiverLaboratories) were subcutaneously inoculated in the right lateral flankwith 10×10⁶ A549 shRNA cells in 100 μl HBSS/MATRIGEL® (BD Biosciences)or with 10×10⁶ H441 shRNA cells in 100 μl HBSS per mouse. When tumorvolume reached approximately 150-250 mm³, mice were randomized toreceive drinking water containing 1 mg/ml doxycycline (in 5% sucrose) orno doxycycline (5% sucrose alone) adlibitum. The doxycycline wasreplaced 3 times a week and the sucrose replaced once a week. Tumorvolumes were determined using digital calipers (Fred V. Fowler Company,Inc.) using the formula (L×W×W)/2 and plotted as mean tumor volume(mm³)+/−SEM. Tumor growth inhibition (% TGI) was calculated as thepercentage of the area under the fitted curve (AUC) for the respectivedose group per day in relation to the vehicle, such that %TGI=100×1−(AUC treatment/day)/(AUC vehicle/day). In a separate study,mice with 150-250 mm³ tumors were dosed with 1 mg/ml doxycycline for 5days before the tumors were excised and analyzed by Western blotting forNRF2 levels.

L. A549 Xenografts Treated with ErbB3 Antibodies

Female nude mice (n=10) bearing subcutaneous A549 tumors (75-144 mm3) onDay 1 were treated with vehicle or 50 mg/kg YW57.88.5 (100 mg/kg loadingdose) administered intravenously once each week for four weeks (qwk×4).Tumors were measured twice each week, and each animal was euthanized forendpoint at the earlier of its tumor reaching a volume of 1000 mm³ or onthe final day of the treatment regimen.

Example 2: Identification of NSCLC Cell Lines with Mutations in KEAP1and NRF2

To identify mutations, copy number, and loss of heterozygosity (LOH) ofKEAP1 and NRF2 in NSCLC, a panel of 113 NSCLC cell lines profiled byRNA-seq, exome-seq, or SNP arrays was documented (FIG. 1A). KEAP1mutations were found in 29/113 cell lines (26%), and NRF2 mutations weredetected in 4/113 cell lines (4%). Except for the NCI-H661 cell line,all KEAP1 mutated cell lines showed homozygous expression of the mutatedallele, which was generally associated with copy neutral LOH. Incontrast, the NRF2 mutations were heterozygous and not associated withLOH. A further two cell lines (HCC1534 and NCI-H1437) showed nodetectable KEAP1 mRNA through bi-allelic loss of KEAP1 DNA. The NRF2mutations were in the previously identified hotspots in the KEAP1interface regions (FIG. 1B) (Shibata et al. Proc. Natl. Acad. Sci.U.S.A. 105:13568-13573, 2008), and comprised point mutations and anin-frame 3-amino-acid deletion. The mutations in KEAP1 were spreadthroughout the primary sequence (FIG. 1C), with few obvious hotspots.However, when mapped onto the KEAP1/NRF2 peptide crystal structure(Fukutomi et al. Mol. Cell. Biol. 34:832-846, 2014), the mutationscluster in the loops extending from the KEAP1 core beta propeller closeto the interaction site with NRF2 (FIG. 1D).

Example 3: Identification of a Mutant KEAP1 Gene Signature

To determine the transcriptional consequences of KEAP1 mutations inNSCLC cell lines, genes that were significantly differentially expressed(p<0.01, absolute mean fold-change >2) in the KEAP1 mutated cell linescompared to the wild-type KEAP1 cell lines were identified. Overall, 27genes were significantly up-regulated in the KEAP1 mutant cell lines(FIGS. 2A-2B), 15 of which have previously been identified as NRF2target genes from ChIP-seq or RNA-seq studies (Chorley et al. NucleicAcids Res. 4:7416-7429, 2012; Hirotsu et al. Nucleic Acids Res.40:10228-10239, 2012; Malhotra et al. Nucleic Acids Res. 38:5718-5734,2010). Only one gene, HSPB1, was identified as significantlydown-regulated using these cut-offs.

Unsupervised clustering of 230 TCGA lung adenocarcinomas based on theexpression of these 27 genes resulted in the division of two majorgroups (FIG. 3A). One group was mainly characterized by high expressionof the 27 signature genes, and contained 43 tumors, out of which 32(74%) were KEAP1 mutant. The other group, characterized by lowexpression, contained 187 tumors, out of which 179 were KEAP1 wild-type.Strikingly, using the same gene set to cluster lung squamous cellcarcinomas, NRF2 as well as KEAP1 mutant tumors were distinguished fromthe NRF2/KEAP1 wild-type tumors (FIG. 3B), suggesting that NRF2 mediatesmost of the transcriptional consequences of KEAP1 loss/mutation.Interestingly, there were several squamous NSCLC tumors that showed highexpression of the KEAP1 mutant genes without any known mutations ineither KEAP1 or NRF2. Of the 27 genes up-regulated in the KEAP1 mutantcell lines, proteomic data was available for 17 in a smaller sub-set ofcell lines (37 wild-type KEAP1, 6 mutant KEAP1). Consistent with theincreased levels of mRNA of these genes in the mutant KEAP1 cell lines,the protein targets of all but one of these 17 genes (SLC7A11, which hadlow peptide coverage) also showed increased expression in the mutantKEAP1 cell lines relative to the wild-type cell lines (FIG. 4).

Example 4: Identification of Aberrant Splicing of NRF2 in Tumor Samples

For the majority of tumors with high expression of the 27 candidate NRF2target genes, elevated gene expression could be explained by mutationsin KEAP1 or NRF2. However, there were some tumors that showed highexpression of candidate NRF2 target genes in the absence ofcharacterized mutations in either KEAP1 or NRF2. Cancer-associatedtranscript alterations are increasingly recognized as possible driverevents. Therefore, it was hypothesized that NRF2 pathway activation inthese tumors might be driven by splice alterations not recognized bywhole-exome sequencing. 54 known oncogenes were analyzed to identifysplice variants that are recurrently observed in cancer samples from theTCGA but are rarely detected in normal samples from the GTEx (seeExample 1). 19 cancer types were selected, each including at least 100cancer samples (6,359 samples in total). In the 54 considered oncogenes,nine recurrent candidate cancer-specific splice variants were identified(2 samples and >1% of samples for a given cancer type). Using the samedetection criteria as in the cancer samples, none of these variantscould be detected in normal controls (2,958 samples in total). Groupingtogether related variants with shared splice sites yielded fiveindependent alterations in four oncogenes (FIG. 5). These alterationsincluded several well-documented oncogenic splice variants, includingEGFRvIII in brain cancers, MET exon 14 skipping in lung adenocarcinomaand CTNNB1 exon 3 deletions in colorectal cancers (Cho et al. CancerRes. 71(24):7587-7596, 2011; Kong-Beltran et al. Cancer Res.66(1):283-289, 2006; Iwao et al. Cancer Res. 58(5):1021-1026, 1998).Interestingly, previously uncharacterized splice variants in NRF2 wereobserved and occurred frequently in patients with squamous NSCLC (3.3%;16/481) and at lower prevalence in patients with HNSC (1.5%; 6/403)(FIG. 5A). A more detailed analysis of NRF2 splice variants in lungsquamous carcinoma revealed two splice variants co-occurring in the samepatients, corresponding to a skip of NRF2 exon 2 in mRNAs transcribedfrom either one of two alternative promoters (2.1%; 10/481) (FIG. 6).Two additional splice variants co-occurred in a distinct set of patients(1.2%; 6/481), corresponding to a skip of both NRF2 exons 2 and 3 (exon2+3) in mRNAs with either one of the two alternative transcript starts(FIG. 6). All patients expressing NRF2 splice variants lacking exon 2 orexon 2+3 also showed expression of normal NRF2 transcripts as evidencedby split reads supporting inclusion of exon 2. Both exons 2 and 3 arepart of the NRF2 coding sequence, and skip of exon 2 or exon 2+3 arepredicted to result in protein isoforms with either an N-terminaltruncation or an in-frame deletion (FIG. 7). The high recurrence of NRF2transcripts lacking exon 2 and preservation of coding potential suggestthat these splice variants may present gain-of-function eventsconferring a selective advantage. This is supported by the finding thatexon 2 encodes the Neh2 domain, which allows for interaction with KEAP1(Itoh et al. Genes Dev. 13(1):76-86, 1999), which is mutated in 15% ofsquamous lung cancers.

To assess whether the observed NRF2 splice variants can account for NRF2pathway activation in patients without mutations in KEAP1 or NRF2,co-occurrence of NRF2 splice variants and NRF2 pathway mutations wereobserved. In the TCGA collection, 178 of the squamous lung tumors wereprofiled by exome-seq. In this subset, 10 tumors (6%) displaying exon 2or exon 2+3 deletion were mutually exclusive with 48 tumors (27%)showing mutations in either NRF2 or KEAP1 (FIG. 8A). Moreover, allexon-2 deleted tumors showed high expression of the 27 candidate NRF2target genes (FIG. 8B). Similar observations were made for head and neckcancer, where NRF2 exon deletion in 5 tumors (2%) were mutuallyexclusive with mutations in NRF2 or KEAP1 in 26 tumors (9%) (FIGS.9A-9B). These results suggest that deletion of exon 2 represents analternative mechanism for activation of NRF2 in a subset of squamousNSCLC and head and neck tumors. Importantly, these results show thatconsideration of splice alterations, in addition to exome sequencing,increased the percentage of patients identified as having putative NRF2pathway activation from 27% (48/178) to 33% (58/178) in lung squamouscarcinoma and from 9% (26/275) to 11% (31/275) in head and neck squamouscarcinoma.

Example 5: Validation of NRF2 Splicing Defects in Cell Lines

To identify cell line models for further study, read evidence for theidentified splice variants in RNA-seq data was analyzed from a largepanel of human cancer cell lines (described in Klijn et al. Nat.Biotechnol. 33(3):306-312, 2014). Out of 611 cell lines, one multiplemyeloma cell line KMS-27 and one hepatocellular carcinoma cell lineJHH-6 were identified, both showing evidence for heterozygous skip ofNRF2 exon 2 by junction reads (FIG. 10). The NRF2 exon 2 skipping byRT-PCR in JHH-6 and KMS-27 mRNA was validated. Using a series of forwardand reverse primers derived from exon 1 and exons 3/4 respectively (FIG.11A), the exon 2 deletion (Δe2 NRF2) in mRNA isolated from JHH-6 andKMS-27 cells was confirmed (FIG. 11B). Sequencing of the PCR productsconfirmed the expected deletion of exon 2 (FIGS. 12A-12C). Based onRNA-seq data no point mutations were detected in the coding sequence ofNRF2 or KEAP1 in JHH-6 or KMS-27 (Klijn et al. Nat. Biotechnol.33(3):306-312, 2014).

As NRF2/KEAP1 alterations are fairly common in hepatocellular carcinoma(10%) but infrequent in multiple myeloma (0%), JHH-6 cells were furthertested. Specifically, expression of the exon 2-deleted form of NRF2protein was tested. Western blotting of whole-cell lysates from JHH-6cells, as well as the KEAP1 mutant HUH-1 line, and HuCCT1 cells as arepresentative wild-type KEAP1 liver cancer cell line was performed. Thelevels of NRF2 in JHH-6 cells were comparable to those seen in HUH-1cells, which were much higher than in the wild-type KEAP1 HuCCT1 cells(FIG. 13). Moreover, a smaller molecular weight species, consistent witha deletion of exon 2, was detectable in JHH-6 and was reduced uponNFE2L2 siRNA transfection, confirming that it indeed represents a formof NRF2. While the altered NRF2 isoform was visible, it was surprisingthat it was not more abundant, given the lack of KEAP1 interactionmotifs. It was hypothesized that a phosphorylated form of exon 2-deletedNRF2 might co-migrate with the unphosphorylated form of wild-type NRF2in the 4-12% gels used. Indeed, dephosphorylation of JHH-6 lysatesshowed that the exon 2-deleted form of NRF2 was significantly moreabundant than the wild-type form (FIG. 14A, middle panel). Similarly,KMS-27 cells expressed the exon 2-deleted form of NRF2, which was themajor species apparent upon dephosphorylation (FIG. 15).

The stability of NRF2 in the three liver cancer cell lines was testedusing cycloheximide to abolish total protein synthesis. Dephosphoryaltedlysates were used to allow more accurate quantification of total NRF2.The experiments showed increased stability of Δe2 NRF2 in JHH-6 cells,comparable to NRF2 in HUH-1 cells, which were both more stable than NRF2in HuCCT1 cells (FIGS. 14A-14B). The exon 2-deleted form of NRF2 inJHH-6 cells also showed prominent nuclear localization, also whencompared to HUH-1 cells (FIG. 16).

To determine whether the deletion of exon 2 in JHH-6 cells made NRF2refractory to regulation by KEAP1, the stability of NRF2 in response toKEAP1 knockdown was tested. Knockdown of KEAP1 in HuCCT1 cells resultedin increased steady state levels of NRF2 due to increased stability(FIG. 14C). However, knockdown of KEAP1 in JHH-6 cells did not affectthe levels or stability of exon 2-deleted NRF2. As expected, knockdownof KEAP1 did not increase the stability of wild-type NRF2 in the KEAP1mutant HUH-1 cell line (FIG. 14D).

Example 6: Assessment of Exon 2 and/or Exon 2+3 Deletion on NRF2

Utilizing the NRF2/KEAP1 gene signature described in Example 3, it wasdetermined that, of 16 hepatocellular carcinoma cell lines, JHH-6 cellsshow among the highest expression of NRF2 target genes from RNA-seqdata, similar to those seen in mutant KEAP1 expressing lines (FIG. 17A).Similarly, out of 18 multiple myeloma cell lines examined, KMS-27 cellsshow among the highest expression of these genes (FIG. 17B). Expressionof these genes can be summarized by a “NRF2 target gene score”calculated as the mean of z-scores for individual target genes acrossthe 611 cell lines examined. This results in a single score per cellline that reflects the extent of overexpression of signature genes inthe given line. The NRF2 target score confirms that JHH-6 cells show asimilar score as liver cancer cell lines expressing KEAP1 mutations(FIG. 18A) and KMS-27 cells show the highest score among multiplemyeloma cell lines (FIG. 18B), despite multiple myeloma showing a lowoverall NRF2 target gene score (indicated by the negative values).

Next, the dependence of JHH-6 cells expressing exon 2 deleted NRF2 onthe expression of NRF2 protein was compared to wild-type NRF2 expressingHuCCT1 cell. Knockdown of NRF2 in JHH-6 cells caused a marked decreasein cell viability, similar to that seen in the mutant KEAP1hepatocellular carcinoma cell line HUH-1. In contrast, NRF2 knockdownhad a more modest effect on the viability of HuCCT1 cells (FIG. 19).This was not due to defective NRF2 knockdown in HuCCT1 cells, as NRF2knockdown was equally efficient in all three cell lines (FIG. 20).Knockdown of NRF2 also resulted in decreased expression of fourwell-characterized NRF2 target genes, although this was slightly reducedin the wild-type KEAP1 HuCCT1 cell line (FIG. 21). Decreased viabilitywas likely due, at least in part, to apoptosis as measured by anincrease in fragmented DNA (FIG. 22).

To address how loss of NRF2 exon 2 affects the ability of NRF2 to beregulated by KEAP1, transient expression in 293 cells was used. KEAP1decreased the expression of full-length NRF2, but had lesser effects onthe expression of NRF2 lacking exon 2 or exons 2+3 (FIG. 23, upperpanels). The inhibitory effect of KEAP1 on full-length NRF2 expressionwas mostly abolished by proteasome inhibitor MG132, as expected.Full-length NRF2 and KEAP1 interacted with each other, whereas deletionof exon 2 or exon 2+3 completely abolished the ability of KEAP1 to bindNRF2 (FIG. 23, lower panels). As a result, truncated NRF2 remainedstable following KEAP1 expression, in contrast to wild-type NRF2 (FIGS.24A-24B), although the truncated forms of NRF2 appeared to have slightlydecreased intrinsic stability. However, altered NRF2 isoforms weretranscriptionally active, as judged by their ability to increase NRF2target gene expression (FIG. 25). Most genes were similarly increased byexon 2- or exon 2+3-deleted NRF2 compared to full-length NRF2 and wereresistant to the effects of KEAP1 overexpression. Interestingly, exon2+3-deleted NRF2 was defective for increasing GPX2 expression,suggesting that there might be subtle differences in the transcriptionalactivation of this form of NRF2. Consistent with this observation, 22 ofthe 27 target genes described in Example 3, in addition to GPX2, showedlower median expression in exon 2+3-deleted squamous lung tumorscompared to exon 2-deleted tumors (FIG. 26).

Example 7: Mechanistic Analysis of NRF2 Exon 2 Splice Alteration

Analysis of exome-seq data for KMS-27 and JHH-6 shows a decrease inreads mapping to exon 2, suggesting that the observed transcriptvariants could be the result of genomic alterations (FIG. 27A).Whole-genome sequencing (WGS) of JHH-6 and KMS-27 showed that these celllines harbor microdeletions surrounding NRF2 exon 2, spanning 4,685 and2,981 nucleotides, respectively (FIG. 27B). To investigate the causalmechanism in patients, targeted paired-end exome-seq data from a largecohort (n=1,218) of clinical squamous NSCLC tumors with high readcoverage (>300×) were analyzed. In this data set, eleven tumors showed adecrease in copy number for exon 2 or exons 2+3 compared to nearbycontrol regions (Materials and Methods; FIG. 27B). The focal nature ofthe deletions can be appreciated by investigating log-ratios fromdefined genomic regions targeted for sequencing (FIGS. 28B-1 and 28B-2).Seven tumors with discordant read pairs were consistent with structuralvariants encompassing several kilobases of DNA and affecting exon 2 orexon 2+3 (FIG. 28A). In total, sixteen patients showed evidence forgenomic alterations affecting NRF2 exon 2 or exon 2+3, and theidentified events were mutually exclusive with point mutations or indelsin NRF2 and KEAP1, which are known to activate this pathway. Anadditional cohort of 45 squamous NSCLC tumors were analyzed, for whichboth RNA and DNA were available. RT-PCR analysis identified a singlepatient with loss of exon 2, which was strongly enriched in the tumorcompared to adjacent normal tissue (FIG. 29). RNA-seq analysis confirmedthat the transcript variant was expressed in the identified tumor, butwas absent in adjacent normal tissue (FIG. 30). Expression of NRF2target genes was also elevated to a similar extent as in TCGA tumorswith known mutations in this pathway, whereas the adjacent normal tissueshowed low expression of these genes (FIG. 31). Finally, whole-genomesequencing confirmed that the transcript variant was the result of asomatic genomic microdeletion of 5,233 nucleotides surrounding exon 2(FIG. 28C). These data suggest that genomic microdeletions are aclinically relevant mechanism for NRF2 pathway activation.

These data suggest that the set of genes regulated by NRF2 is conservedacross different tissues and conditions. This has practical value in theuse of a single gene signature to identify tumors with NRF2 activationin both NSCLC and HNSC (FIG. 32). Interestingly, this NRF2/KEAP1signature is only activated in tumors. Matched normal samples for lungand head and neck tumors showed only low NRF2 target gene activity (FIG.33). This suggests that inhibition of the NRF2 pathway might haveselective benefit in tumors showing pathway deregulation compared tonormal tissues.

Intragenic genomic deletions that result in activation ofproto-oncogenes have previously been reported for a number of genes,including EGFR and CTNNB1. Such variants are not routinely assayed, duein part to limitations of current genomic technologies. In particular,small aberrations affecting individual exons and involving small copynumber changes are difficult to detect by exome-seq alone. Thus,intragenic deletions have remained relatively unexplored and newvariants are still being discovered. Recent studies of small cell lungcancer and adult T cell leukemia/lymphoma identified recurrentmicrodeletions in TP73, IKZF2, and CARD11 using whole-genome sequencing(George et al. Nature 524, 47-53:2015; Kataoka et al. Nat. Genet.47:1304-1315, 2015). In the present study, publicly available RNA-seqdata generated as part of the TCGA project was used to identifyrecurrent transcript alterations in known oncogenes. Due to differencesbetween patient cohorts, it is difficult to assess the generalprevalence of NRF2 exon deletions. For example, when analyzing TCGA lungsquamous cancers with available RNA-seq data (n=481), we identified 3%(16/481) of patients having a deletion of NRF2 exon 2 or exon 2+3. Whenanalyzing the subset of patients with available exome-seq data (n=178),for which somatic mutation calling can be performed, the proportion ofpatients with NRF2 exon deletions was 6% (10/178). Accounting for NRF2exon deletions increased the percentage of patients with putative NRF2pathway activation from 27% (48/178) to 33% (58/178) in lung squamouscarcinoma and from 9% (26/275) to 11% (31/275) in head and neck squamouscarcinoma, compared to assessing mutations in NRF2 or KEAP1 by exome-seqalone (FIGS. 8A and 9A). Analysis of real-world clinical samples frompatients that underwent genomic profiling suggested a prevalence of NRF2exon deletions in 1-2% of lung squamous cell carcinoma. However, thelatter analysis lacks sensitivity since optimized criteria fordetermining single-exon deletions in samples with variable tumor contenthave yet to be established and only unambiguous deletions wereconsidered. Nevertheless, the results presented herein are consistentwith the concept that modulation of this pathway is frequently alteredin specific tumor indications, such as squamous NSCLC and head and neckcarcinomas. Additional screening of known cancer genes throughsequencing of complete gene loci, including introns, or by combiningdata from exome and RNA sequencing experiments may also be performed.

Analysis of the structure of the three deletions identified by WGSshowed that breakpoints were distinct, but in each case genomic regionsflanking the deletions showed 2-6 nucleotides with sequence homology(FIG. 34). The DNA sequences of the 3′ end, 5′ end, and junction read ofJHH-6 cells are provided by SEQ ID NOs: 61-63, respectively. The DNAsequences of the 3′ end, 5′ end, and junction read of KMS-27 cells areprovided by SEQ ID NOs: 64-66, respectively. The DNA sequences of the 3′end, 5′ end, and junction read of primary tumor cells are provided bySEQ ID NOs: 67-69, respectively.

NRF2 often shows genomic amplification in addition to point mutations.Interestingly, while the intensity of the NRF2 deletion product inKMS-27 cells by RT-PCR analysis appeared similar to wild-type NRF2, itseemed to be more abundant in JHH-6 cells (FIG. 13). This was alsoreflected in WGS read counts, which suggested a higher abundance of thedeleted form compared to the wild-type allele (FIG. 27B). These resultsare consistent with the observation that JHH-6 cells carry five copiesof the NRF2 gene locus by SNP array, whereas KMS-27 cells carry twocopies. Amplification of NRF2 is reasonably frequent in the TCGA samplesanalyzed, including squamous (4.5%) and adenomatous (2.6%) NSCLC, HNSC(12.2%), and liver cancers (3.6%), and represents a mechanism toincrease NRF2 transcriptional output. In the case of JHH-6 cells, thesedata suggest that the deleted allele has been preferentially amplified,providing an additional mechanism to boost NRF2 signaling in this cellline. However, preferential amplification of the truncated/splicedallele was not observed in the primary tumors, suggesting that exon 2 or2+3 deletion alone can provide sufficient NRF2 activity for clonalselection.

Deletion of exon 2 provides an elegant mechanism to increase NRF2activity by removing the interaction site with KEAP1, while keeping theremainder of the gene functionally intact for DNA binding andtranscriptional activation functions. Indeed, our biochemical analysesconfirmed the almost complete loss of KEAP1 binding and resultingstabilization of NRF2 when exon 2 is deleted (FIGS. 23 and 24). Whenconsidering NRF2 point mutations found in tumors, mutations surroundingthe ETGE high-affinity binding site result in complete loss of KEAP1interaction, whereas mutations in the lower affinity DLG motif vary intheir ability to disrupt the NRF2/KEAP1 complex (Fukutomi et al. MolCell Biol. 34(5):832-846, 2014; Shibata et al. Proc. Natl. Acad. Sci.USA. 105(36):13568-13573, 2008). However, even point mutations that donot disrupt the complex change the nature of the interaction such as toprevent KEAP1-mediated ubiquitination of NRF2 (Shibata et al. Proc.Natl. Acad. Sci. USA. 105(36):13568-13573, 2008). While the interactionwith KEAP1 is similarly abolished in the case of deletion of both exon 2and 3, exon 3 contains the Neh4 domain that has been previouslyimplicated in transcriptional activation by NRF2 through binding to CREB(cAMP Responsive Element Binding protein) Binding Protein (CBP) (Katohet al. Genes Cells. 6(10):857-868, 2001). Neh4 (contained in exon 3) andNeh5 (contained in exon 4) were shown to act synergistically inrecruiting CBP. Consistent with this, a decreased ability of Δe2+3 NRF2to induce some NRF2 target genes compared to Δe2 NRF2 ortumor-associated point mutations in NRF2 was observed (FIGS. 25 and 26).

Deletions found in human tumors that remove the interaction domain withE3 ligases have also been observed in other genes. For example, 7 out of222 colorectal tumors showed small genomic deletions (234-677 bp)surrounding exon 3 of β-catenin (Iwao et al. Cancer Res.58(5):1021-1026, 1998) that removes the interaction site for its E3ligase β-TRCP (Hart et al. Curr. Biol. 9(4):207-210, 1999). Similarly,the majority of TMPRSS-ERG fusion proteins found in prostate cancerencode truncated versions of ERG that render them resistant toubiquitination and degradation mediated by SPOP (An et al. Mol. Cell.59(6):904-916, 2015).

In addition, mutations resulting in MET exon 14 skipping remove aminoacid residue Y1003, which is required for Cbl recruitment and subsequentubiquitination and down-regulation. Therefore, small intragenicdeletions represent effective mechanisms for nascent oncogenes to escapenormal degradation during tumor initiation and evolution.

Example 8: NRF2 Knockdown in Mutant KEAP1 Cells

This example provides a characterization of the effects of KEAP1mutations on the requirement for NRF2 activity under different growthenvironments and shows that NRF2 activity is essential for growth inanchorage independent conditions.

The consequences of NRF2 inhibition across wild-type and mutant KEAP1and NRF2 cell lines were examined. Stable cell lines expressing threeindependent NRF2 shRNAs under the control of doxycycline, as well asthree independent non-targeting controls (NTCs) were established. TheseNRF2 shRNAs were effective at reducing NRF2 protein levels in five KEAP1mutant cell lines, two NRF2 mutant cell lines, and five wild-type NSCLCcell lines, as well as in immortalized but non-transformed lungepithelial BEAS2B cells (FIG. 35). Upon doxycycline addition, viabilityof most cell lines was decreased to varying extents, with the KEAP1mutant cell lines generally exhibiting significantly greater decreases(FIGS. 36 and 37). Knockdown of NRF2 by siRNA in a larger panel of NSCLCcell lines confirmed a genotype-dependent effect on cell viability (FIG.38).

The consequence of NRF2 knockdown in tumor xenografts was characterized.The KEAP1 mutant A549 cell line and the KEAP1 wild-type H441 cell linesexpressing dox-inducible NRF2 shRNAs were implanted into the flanks offemale SCID mice. NRF2 was effectively knocked down in doxycyclinetreated mice in both tumors (FIGS. 39 and 40). NRF2 knockdown in theKEAP1 mutant A549 cell line had a dramatic effect on tumor growth,resulting in complete tumor regression in 5 out of 10 tumors (FIG. 41A).In contrast, the effect on KEAP1 wild-type H441 growth was more modest,resulting in a 37% reduction in tumor growth with all animals displayingmaintained tumor burden (FIG. 41B).

To understand the differential effects between NRF2 knockdown on tumorpropagation in xenografts versus 2D growth on plastic, severaladditional cell culture environments were tested. NRF2 knockdown incells grown in low adherence plates and/or low oxygen (0.5%) showedsimilar consequences to cells grown on plastic (FIG. 42). In contrast,the growth of KEAP1 mutant cell lines was severely compromised whencultured in soft agar (FIGS. 43 and 44), on micropatterned plastic films(FIGS. 45 and 46), or in methylcellulose (FIG. 47). Growth in soft agarwas used to characterize the consequences of NRF2 knockdown in moredetail. While knockdown of NRF2 completely abolished colony formation inthree KEAP1 mutant cell lines, it had almost no effect in H1048 andH441, two wild-type KEAP1 NSCLC cell lines (FIGS. 43 and 44). The roleof the glutathione pathway in the response to NRF2 knockdown wasassessed, as this pathway has been shown to mediate survival propertiesfacilitated by high NRF2 activity. While addition of reduced glutathionegenerally increased the ability of all tested cell lines to formcolonies in soft agar, it was unable to rescue the consequences of NRF2knockdown (FIGS. 43 and 44). Similar negative results were seen withN-acetyl cysteine (NAC; FIG. 48). Exogenous glutathione was able toenter cells and reduce reactive oxygen (ROS) levels, as measured bydichlorofluorescein staining (FIG. 49). Thus, the requirement for NRF2activity is surprisingly independent of the glutathione synthesispathway.

To further explore the effects of the glutathione pathway in NRF2responses, the expression and activity of the xCT glutamate/cysteineantiporter, one of the rate limiting steps in glutathione synthesis, wasmonitored. SLC7A11 expression was reduced following NRF2 knockdown (FIG.50), causing a decrease in cystine uptake (FIG. 51) associated withreduced glutathione (FIG. 52). NRF2 knockdown also caused a largeincrease in ROS levels (FIG. 53). To determine whether inhibition ofSLC7A11 expression and cystine uptake contributed to decreased viabilityfollowing NRF2 knockdown, xCT function was initiated using erastin,which inhibited cystine uptake (FIG. 51) and increased oxidative stress(FIG. 53). However, this was not sufficient to decrease the viability ofthe KEAP1 mutant cell line A549 (FIG. 54) or most other KEAP1 mutantcell lines (FIG. 55). The combination of erastin and NRF2 knockdown,however, did result in a dramatic decrease in viability (FIG. 54).Similarly, the glutathione synthase inhibitor buthionine sylphoximine(BSO) or the glutaminase inhibitor BPTES also did not displaypreferential toxicity for KEAP1 mutant cell lines (FIGS. 56 and 57).These results indicate that supplementation with glutathione was notsufficient to rescue lethality induced by NRF2 knockdown, nor wasdepletion of glutathione sufficient to kill KEAP1 mutant cell lines.

In order to understand which pathways were activated as a consequence ofNRF2 activation or KEAP1 loss, a CRISPR screen was performed using alibrary of genes that were decreased upon NRF2 knockdown in A549 cellsand/or elevated in a panel of KEAP1 mutant NSCLC cell lines. As distinctconsequences were observed following NRF2 knockdown in 2D, 3D, andxenograft growth conditions, the screen was performed under all threeenvironments to determine whether discrete dependencies could beidentified. At 15-day time point for all three conditions, all threescreens performed similarly, with gRNAs representing only a small numberof genes showing significant drop-out (FIGS. 58-60). NFE2L2 and itsbinding partner, MAFG, were among the most significant genes, showingthat the screen performed as expected. The pentose phosphate pathwaygenes PGD, G6PD and TKT, known NRF2 target genes, also showed strongdrop-out. Other strong hits in the screen were two growth factorreceptor genes, IGF1R and ERBB3, and genes encoding three components ofa redox signaling relay, PRDX1, TXN, and TXNRD1.

Expression of ErbB3 was decreased following NRF2 knockdown in A549 cells(FIGS. 58-60). Treatment with YW57.88.5 in a tumor xenograft modelindicated that ErbB3 was required for A549 proliferation (FIG. 61).

Expression of IGF1R was greater in KEAP1 mutant NSCLC cells relative toKEAP1 wild-type NSCLC cell lines. To test the effect of IGF1R inhibitionon KEAP1 mutant and KEAP1 wild-type cells, cell lines were treated withlinsitinib, a potent and selective IGF1R small molecule inhibitor.Linsitinib showed little effect on proliferation when tested in threewild-type and three mutant KEAP1 NSCLC cell lines. However, thiscompound was very potent at inhibiting colony growth of A549 cells insoft agar, having an IC₅₀ of about 20 nM. Moreover, when tested againsta large panel of NSCLC cell lines, there appeared to be a selectivegrowth inhibition in soft agar of this compound in KEAP1 mutant celllines. A similar selective effect on KEAP1 mutant cell lines when grownunder anchorage independent conditions was also seen with an independentIGF1R inhibitor NVP-AEW541 (FIG. 62).

Thus, growth factors signaling through IGF1R and ErbB3 are significantmediators of the growth of KEAP1 mutant cells.

OTHER EMBODIMENTS

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. A method of diagnosing a cancer in a subject, the method comprising:(a) determining the expression levels of the following 27 genes:AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR,SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH,TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sampleobtained from the subject; and (b) comparing the expression level ofeach of the 27 genes to a reference expression level of each of the 27genes, wherein an increase in the expression level of each of the 27genes in the sample relative to the respective reference expressionlevel of each of the 27 genes identifies the subject having a cancer. 2.The method of claim 1, wherein the method further comprises: determiningif the subject's cancer is a NRF2-dependent cancer, wherein an increasein the expression level of each of the 27 genes in the sample relativeto the respective reference expression level of each of the 27 genesidentifies the subject as having a NRF2-dependent cancer. 3-8.(canceled)
 9. The method of claim 1, wherein (a) the expression level ofeach of the 27 genes in the sample is an average expression level ofeach of the 27 genes of the sample; (b) the reference expression levelof each of the 27 genes is an average expression level of each of the 27genes of the reference; and (c) the average expression level of each ofthe 27 genes of the sample is compared to the average of each of the 27genes of the reference. 10-11. (canceled)
 12. The method of claim 1,wherein the reference expression level of each of the 27 genes is themean level of expression of each of the 27 genes in a population ofsubjects having the cancer.
 13. The method of claim 12, wherein thereference expression level is the mean level of expression of each ofthe 27 genes in a population of subjects having lung cancer, optionallya non-small cell lung cancer (NSCLC), optionally a squamous NSCLC.14-15. (canceled)
 16. The method of claim 1, wherein the expressionlevel is an mRNA expression level, optionally wherein the mRNAexpression level is determined by PCR, RT-PCR, RNA-seq, gene expressionprofiling, serial analysis of gene expression, or microarray analysis.17. (canceled)
 18. The method of claim 1, wherein the expression levelis a protein expression level, optionally wherein the protein expressionlevel is determined by western blot, immunohistochemistry, or massspectrometry.
 19. (canceled)
 20. The method of claim 1, furthercomprising determining a DNA sequence of NRF2, optionally wherein theDNA sequence is determined by PCR, exome-seq, microarray analysis, orwhole genome sequencing. 21-32. (canceled)
 33. The method of claim 1,further comprising administering to the subject a therapeuticallyeffective amount of a NRF2 pathway antagonist and/or a therapeuticallyeffective amount of an anti-cancer agent. 34-40. (canceled)
 41. A methodof treating a subject having a cancer, the method comprisingadministering to the subject a therapeutically effective amount of aNRF2 pathway antagonist, wherein the expression level of each of thefollowing 27 genes AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16,ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2,AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, andFTL in a sample obtained from the subject has been determined to beincreased relative to a respective reference expression level of each ofthe 27 genes. 42-47. (canceled)
 48. The method of claim 41, wherein (a)the expression level of each of the 27 genes in the sample is an averageexpression level of each of the 27 genes of the sample; (b) thereference expression level of each of the 27 genes is an averageexpression level of each of the 27 genes of the reference; and (c) theaverage expression level of each of the 27 genes of the sample iscompared to the average of each of the 27 genes of the reference. 49-50.(canceled)
 51. The method of claim 41, wherein the reference expressionlevel is the mean level of expression of each of the 27 genes in apopulation of subjects having the cancer, optionally lung cancer,optionally NSCLC, optionally squamous NSCLC. 52-53. (canceled)
 54. Themethod of claim 41, wherein the expression level is an mRNA expressionlevel, optionally wherein the mRNA expression level is determined byPCR, RT-PCR, RNA-seq, gene expression profiling, serial analysis of geneexpression, or microarray analysis. 55-56. (canceled)
 57. The method ofclaim 41, wherein the expression level is a protein expression level,optionally wherein the protein expression is determined by western blot,immunohistochemistry, or mass spectrometry.
 58. (canceled)
 59. Themethod of claim 41, further comprising determining a DNA sequence of theNRF2, optionally wherein the DNA sequence is determined by PCR,exome-seq, microarray analysis, or whole genome sequencing. 60-78.(canceled)
 79. The method of claim 1, wherein the sample obtained fromthe subject is from a biopsy sample.
 80. (canceled)
 81. The method ofclaim 1, wherein the subject: (a) is a previously untreated subject;and/or (b) has a lung cancer or a head and neck cancer.
 82. The methodof claim 81, wherein the lung cancer is a non-small cell lung cancer(NSCLC).
 83. The method of claim 82, wherein the NSCLC is a squamousNSCLC.
 84. The method of claim 81, wherein the head and neck cancer is asquamous head and neck cancer.